'  EXPERIMENTAL 
DAIRY  BACTERIOLOGY 


BY 


H.  L  RUSSELL,  PH.D. 
v» 


DEAN   OF  THE   COLLEGE   OF  AGRICULTURE 
UNIVERSITY  OF  WISCONSIN 


AND 


E.  G.  HASTINGS,  M.S. 

ASSISTANT   PROFESSOR  OF  AGRICULTURAL   BACTERIOLOGY 
UNIVERSITY  OF  WISCONSIN 


9f THE 

UNIVERSITY 

Of 


GINN  AND  COMPANY 

BOSTON  •  NEW  Y<  >KK  •  CHICAGO  •  JXJNDON 


«* 


COPYKIGHT,   1909,   BY 

H.  L.  RUSSELL  AND  E.  G.  HASTINGS 


ALL  RIGHTS  KESEBVED 


gtfienaum 


GINN  AND  COMPANY  •  PRO- 
PRIETORS •  BOSTON  •  U.S.A. 


INTRODUCTION 

The  purpose  of  the  course  here  outlined  is  to  train  the 
student  in  those  bacteriological  processes  that  are  necessary 
for  him  to  comprehend  thoroughly,  before  he  is  in  a  position 
to  appreciate  the  relation  of  microorganisms  to  dairy  processes. 
This  work  is  of  fundamental  importance  to  the  student  who 
wishes  to  learn  the  nature  of  the  biological  changes  going  on 
in  milk  and  its  products,  whether  he  is  concerned  purely  with 
the  practical  side  of  dairying  or  is  interested  in  the  cognate 
work  of  dairy  chemistry  or  dairy  bacteriology. 

The  attempt  has  been  made  to  keep  the  scope  of  this  work 
within  the  realm  of  dairy  bacteriology,  and  not  encroach  upon 
the  field  of  dairy  manufactures.  For  example,  in  the  study  of 
starters  it  is  desirable  that  the  bacteriological  student  should 
know  how  to  determine  the  purity  and  vigor  of  a  culture,  but 
the  practical  propagation  of  the  starter  should  be  presented 
from  the  creamery  point  of  view.  The  effect  of  the  ripening 
of  cream  on  the  churning  process  and  the  action  of  acid  on  the 
physical  condition  of  cheese  are  instances  of  biologic  activity 
that  can  better  be  studied  in  the  factory  than  in  the  labora- 
tory. In  many  cases  it  would  be  advantageous  if  the  practi- 
cal and  theoretical  work  could  be  carried  on  simultaneously. 
Thus,  in  studying  a  starter  bacteriologically  it  would  be  very 
desirable  to  test  in  the  laboratory  by  frequent  examinations 
the  purity  of  the  starter  as  it  is  handled  in  the  dairy  from 
day  to  day.  The  possibility  of  deterioration  could  then  be 
ascertained  from  both  the  practical  and  scientific  points  of 

iii 

190829 


iv  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

view,  and  each  would  supplement  the  other.  Sucli  studies, 
however,  are  better  adapted  to  the  advanced  student  than  to 
the  beginner,  and  it  is  felt  that  it  is  preferable  to  present  to 
the  elementary  student  the  biologic  aspect  of  the  subject 
more  or  less  completely  divorced  from  the  practical  factory 
work.  The  technique  of  bacteriological  methods  should  be 
mastered  by  each  student  individually.  He  should  learn  how 
to  make  his  media,  and  how  to  care  for  the  same.  After  this 
necessary  technical  foundation  has  been  laid,  it  is  possible  to 
make  certain  experiments  in  groups,  and  thus  economize  in 
time  and  material  with  no  loss  in  pedagogic  value. 

The  methods  presented  are  believed  to  be  the  best  in  use 
at  the  present  time.  A  committee  of  the  American  Public 
Health  Association  now  has  under  consideration  the  formula- 
tion of  standard  methods  for  milk  analysis,  but  these  have  not 
as  yet  been  published.  The  methods  of  media  making  are 
those  recommended  by  the  Laboratory  Section  of  the  Ameri- 
can Public  Health  Association,  and,  while  more  complicated 
than  those  usually  described  in  text-books,  are  surely  more 
desirable  in  establishing  uniform  methods. 

In  bacteriology,  as  in  other  biological  sciences,  unknown 
factors  must  be  dealt  with.  Again,  numerous  conditions  that 
cannot  be  controlled  influence  the  results.  A  long  series  of 
observations  may  be  certain  to  point  in  a  definite  direction, 
while  one  or  two  observations,  such  as  can  be  made  in  a  course 
of  study,  may  give  results  wholly  at  variance  with  what  is 
expected.  This  fact  does  not,  however,  lessen  the  value  of  the 
exercise  in  training  the  student  in  technique  and  in  develop- 
ing his  power  of  observation. 


CONTENTS 

CHAPTER  PAGES 

I.  CULTURAL  TECHNIQUE 1-36 

Glassware 1 

Cleaning  glassware 2 

Sterilization  of  glassware 3 

Preparation  of  media 4 

Ingredients  of  media 5 

Reaction  of  media 5 

Neutralization  of  media .     .  6 

Filtering  of  media 7 

Plugging  tubes 9 

Sterilization  of  media 0 

Care  of  media .     .     <    .     .  11 

Preparation  of  broth 12 

Preparation  of  gelatin 14 

Preparation  of  agar 15 

Preparation  of  milk * 16 

Preparation  of  sugar  media .  17 

Preparation  of  litmus  solution 18 

Preparation  of  water  blanks 18 

Quantitative  analysis  of  milk ..19 

Qualitative  analysis  of  milk 29 

Isolation  of  pure  cultures 30 

Platinum  needles t 30 

Plate  cultures - 31 

Study  of  plate  cultures 32 

Test-tube  cultures 33 

Study  of  test-tube  cultures 35 

II.  MICROSCOPICAL  TECHNIQUE 37-52 

The  microscope  and  its  accessories 37 

Slides  and  cover  glasses 41 

Staining  solutions 42 


vi  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

CHAPTER  PAGES 

Making  of  stained  preparations 44 

Gram's  stain 47 

Flagella  stain 47 

Capsule  stain 48 

Spore  stain 48 

Examination  of  living  bacteria        49 

Hanging  block 51 

Measuring  bacteria 51 

III.  CONTAMINATION  OF  MILK 53-63 

Contamination  from  barn  air 54 

Contamination  from  coat  of  animal 56 

Contamination  from  manure 57 

Contamination  from  hair        58 

Contamination  from  interior  of  udder 58 

Contamination  from  utensils 60 

Washing  utensils 63 

Plan  for  the  production  of  sanitary  milk      . 63 

IV.  MILK  FERMENTATIONS 64-85 

Preliminary  cultivation 65 

Primary  tests  for  identification  of  species 66 

Action  on  carbohydrates 67 

Action  on  nitrates 70 

Formation  of  indol 72 

Relation  to  oxygen 72 

Relation  to  temperature 73 

Chromogenesis 74 

Action  on  milk 74 

Acid  fermentation 76 

Digesting  and  sweet-curdling  fermentation       .     .     .     .     .  77 

Alcoholic  fermentation 78 

Ropy  fermentation 79 

Bitter  fermentation 80 

Cycle  of  fermentations  in  milk 80 

Butyric -acid  fermentation 81 


CONTENTS  vii 

<•  MAI-TEH  PAGES 

V.  PRESERVATION  OF  MILK 86-92 

Effect  of  temperature  on  bacterial  growth 88 

Pasteurization  of  milk       89 

Detection  of  heated  milk.    Storch's  reaction 91 

VI.  RELATION  OF  BACTERIA  TO  BUTTER     .     .     ...       93-102 

Effect  of  creaming  on  the  distribution  of  the  bacteria  in 

milk 93 

Examination  of  cream  and  separator  slime        .     '.     .     .     .  94 

Examination  of  sweet  and  ripened  cream 94 

Relation  of  butter  flavor  to  bacterial  development     ...  95 

Quantitative  analysis  of  butter 95 

Relation  of  age  of  butter  to  bacterial  content 96 

Starters 97 

Relation  of  bacteria  to  keeping  quality  of  butter  .     .     .     .101 

Relation  of  bacteria  in  wash  water  to  butter 101 

VII.  RELATION  OF  BACTERIA  TO  CHEESE  .     .     .     .     .     103-109 

Ripening  of  cheese 103 

Role  of  acid-forming  bacteria     .     .     .    > 104 

Analysis  of  cheese    . 105 

Qualitative  examination  of  milk  for  cheese  making  .     .     .  106 

VIII.  MILK  HYGIENE .     .     .     .     .     110-124 

Milk  as  a  distributer  of  disease       . 110 

Microscopical  examination  for  tubercle  bacilli 112 

Examination  for  tubercle  bacilli  by  animal  inoculation      .   114 
Examination  for  pyogenic  organisms  .     .     .....     .115 

Microscopical  examination  of  milk 116 

Examination  for  leucocytes,  Doane-Buckley  method      .     .117 
I  Examination  for  leucocytes  ;  smeared-sediment  method      .  120 

Examination  for  fibrin 122 

Direct  enumeration  of  bacteria  in  milk 122 

Microscopic  examination  for  streptococci 124 


viii          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

PAGES 
APPENDIX  A.    PREPARATION  OK  PIPETTES 125 

APPENDIX  B.    DESCRIPTIVE  CHART.    SOCIETY  OF  AMERICAN 

BACTERIOLOGISTS 127 

APPENDIX  C.  CONVERSION  FACTORS  FOR  THERMOMETER 
SCALES.  CONVERSION  FACTORS.  METRIC  TO  ENGLISH. 
ENGLISH  TO  METRIC 138 

APPENDIX  D.    APPARATUS  FOR  EACH  STUDENT 139 

GLOSSARY  OF  TERMS 140 

TEXT-BOOKS  ON  GENERAL  AND  DAIRY  BACTERIOLOGY      .     .  143 
INDEX  .  .  145 


EXPERIMENTAL 
DAIRY   BACTERIOLOGY 


CHAPTER   I 

CULTURAL  TECHNIQUE 

Glassware.  In  bacteriological  work  a  large  amount  of 
glassware  must  be  used.  It  is  desirable  to  employ  the  most 
convenient  and  economical  types  of  tubes,  dishes,  etc.  The 
test  tubes  used  for  the  storage  of  media  for  culture  work  are 
not  the  ordinary  thin-walled  test  tubes  of  the  chemical  labo- 
ratory, but  are  made  of  much  heavier  glass  and  without  the 
lip.  Such  tubes  of  thick  glass  are  not  so  easily  broken  in 
handling  and  yet  will  withstand  the  action  of  heat.  A  tube 
120  mm.  long  and  15  mm.  internal  diameter  is  a  convenient 
size,  and  of  sufficient  capacity  for  nearly  all  purposes.  If  it 
is  desirable  to  have  a  tube  holding  10  cc.,  tubes  of  18  mm. 
diameter  will  be  required.  The  tubes  in  use  in  the  laboratory 
should  always  be  of  the  same  length,  120  mm.  or  150  mm. 
The  tube  described  is  usually  known  as  the  "  board  of  health1' 
pattern. 

Petri  dishes  should  have  an  internal  diameter  of  90  mm., 
and  should  be  15  mm.  high ;  the  covers  should  be  10-12  mm. 
deep,  and  should  be  2-3  mm.  greater  in  diameter  than  the 
bottoms.  The  bottoms  of  the  Petri  dishes  should  be  perfectly 
flat.  The  glass  from  which  tubes  and  dishes  are  made  should 

1 


EXPERIMENTAL   DAIRY  BACTERIOLOGY 


withstand  repeated  heating  to  170°  C.  without  flaking  or 
becoming  cloudy,  and  should  be  well  annealed,  so  as  to 
withstand  sudden  changes  in  temperature. 

For  storage  of  media,  and  for  making  the  dilutions  neces- 
sary in  bacteriological  work,  flasks  are  used,  usually  of  the 
Erlenmeyer  form,  with  narrow  necks.  The  flasks  may  be  of 
Jena  glass,  but  the  many  forms  of  "  resistance  glass  "  are  well 
adapted  to  ordinary  purposes,  since  its 
solubility  is  slight.  For  many  purposes, 
as  for  dilution  work,  ordinary  bottles 
may  be  employed ;  they  have  the  ad- 
vantage of  being  cheap  and  economical 
of  space  in  sterilizers  and  elsewhere. 

Cleaning  glassware.  All  glassware 
used  in  the  bacteriological  laboratory 
must  be  perfectly  clean.  New  glass- 
ware must  be  washed  in  hot  water, 
rinsed  in  distilled  or  tap  water,  and 
placed  for  a  short  time  in  1  per  cent 
FIG.  1.  TEST  TUBES  hydrochloric  acid,  to  remove  any  free 

Board  of  health  pattern,      alkali  present  On  the  glass.    After  r ins- 
one-third  size  •          •,    •       i  T          in 

mg,  it  is  drained  and  allowed  to  dry. 

Petri  dishes,  tubes,  etc.,  containing  discarded  cultures,  should 
not  be  allowed  to  become  dry,  but  should  be  washed  as  soon 
as  possible.  The  solid  media  can  be  readily  removed  from 
test  tubes  by  inserting  a  piece  of  glass  tubing,  which  is  at- 
tached to  the  water  tap  by  a  strong  piece  of  rubber  tubing. 
The  tube  is  pushed  to  the  bottom  of  the  test  tube  and  the 
force  of  the  water  ejects  the  solid  matter.  Glassware  con- 
taining whole  milk,  butter,  or  any  fatty  substance  should  not 
be  washed  with  the  ordinary  dishes.  If  the  media  has  been 
allowed  to  become  dry  in  the  tubes  and  plates,  they  must  be 


CULTURAL  TECHNIQUE  3 

boiled  in  water  or  allowed  to  stand  in  a  strong  solution  of 
washing  powder  for  twenty-four  hours.  The  washing  powders 
which  are  so  largely  used  in  dairy  work  will  be  found  prefer- 
able to  soap,  rinsing  from  the  glass  much  easier  and  produc- 
ing no  deposits  of  calcium  salts  with  hard  water. 

Pipettes  which  have  been  used  for  milk  should  be  rinsed 
at  once,  placed  in  a  solution  of  washing  powder  for  several 
hours,  removed,  rinsed  well,  and  drained. 

The  destructive  test-tube  brush  should  be  replaced  by  a 
swab,  made  by  slightly  expanding  the  end  of  a  piece  of 
glass  tubing  and  fastening  to  this,  with  an  ordinary  rubber 
band,  a  bit  of  soft  sponge,  which  may  be  renewed  as  often  as 
necessary. 

Exercise.    Clean  all  test  tubes,  Petri  dishes,  and  flasks  in  your 

possession. 

Sterilization  of  glassware.  Dry  heat  is  not  as  effective  a 
germ  destroyer  as  moist  heat,  but  for  certain  purposes  it  is 
preferable.  It  is  especially  adapted  for  sterilization  of  empty 
culture  dishes,  pipettes,  and  other  glassware.  Organic  matter 
such  as  cotton  must  be  carefully  watched  in  sterilizing  in 
dry  heat,  as  it  chars  when  heated  above  150°  C.  The  slight 
browning  of  the  cotton  plugs  in  test  tubes  is  an  indication  that 
the  heating  has  been  sufficient  to  destroy  all  bacteria  present. 

All  material  that  can  be  subjected  to  dry  heat  is  sterilized 
by  heating  to  150°  C.  for  one  and  one-half  hours  in  a  hot-air 
sterilizer.  The  latter  consists  of  a  double-walled  box  of  sheet 
metal  so  provided  with  burners  as  to  heat  rapidly  and  uni- 
formly. The  glassware  must  be  thoroughly  dry  before  being 
placed  in  the  sterilizer.  In  case  the  Petri  dishes  are  placed 
in  solid  piles,  it  should  be  remembered  that  it  requires  a  con- 
siderable period  of  time  for  the  heat  to  penetrate  into  the 


4  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

inner  dishes;  hence  heating  the  oven  to  175°  C.  and  at  once 
turning  off  the  heat,  as  is  sometimes  recommended,  is  likely 
to  result  in  incomplete  sterilization.  The  sterilizer  should  not 
be  opened  until  it  has  cooled  to  60°  C.  If  cold  air  strikes  the 
hot  glass,  many  dishes  are  likely  to  be  broken. 

After  sterilization  Petri  dishes  should  not  be  opened  until 
used.  They  should  be  handled  as  little  as  possible,  as  mold 
spores  are  likely  to  gain  entrance ;  and  in  case  they  are  to  be 


FIG.  2.    PIPETTE  CASE  AND  PIPETTES 

Case  of  sheet  copper  with  slip  cover ;  pipettes  wrapped  in  tissue  paper,  on  one 
of  which  the  paper  is  torn  to  show  the  cotton  plug  in  the  mouth  end 

carried  from  the  laboratory  or  are  to  be  kept  some  time  before 
using,  they  should  be  wrapped,  before  sterilizing,  in  pieces  of 
newspaper. 

The  mouth  end  of  the  pipettes  should  be  plugged  with 
cotton,  and  the  projecting  fibers  burned  off  so  as  not  to  in- 
terfere in  using  the  pipette.  The  pipettes  should  be  placed 
in  the  metal  case,  points  down,  the  end  of  the  case  plugged 
with  cotton,  and  the  whole  sterilized.  In  place  of  the  metal 
pipette  case  the  pipettes  may  be  prepared  for  sterilization 
by  wrapping  each  in  tissue  paper,  the  paper  at  the  bottom 
being  turned  in  and  the  mouth  end  twisted,  so  that  the 
ends  may  be  distinguished  before  removing  the  wrapper. 

Exercise.    Sterilize  Petri  dishes  and  pipettes. 

Preparation  of  media.  The  media  used  in  the  bacteriologi- 
cal laboratory  bears  the  same  relation  to  bacteriology  as  does 


CULTURAL   TECHNIQUE  5 

the  standard  solution  to  accurate  work  in  chemistry.  It  is 
desirable  that  the  media  shall  be  uniform  in  composition  and 
physical  properties.  This  is,  however,  not  wholly  possible 
with  the  present  methods.  Much  greater  uniformity  than 
usual  might  be  attained,  however,  if  standard  ingredients 
and  uniform  methods  were  insisted  on. 

Ingredients  of  media.  The  following  ingredients  comprise 
those  used  in  the  preparation  of  the  ordinary  media : 

Distilled  water  rather  than  tap  water. 

Infusion  of  meat, —  Liebig's  meat  extract  is  often  used 
as  a  substitute  on  account  of  its  greater  convenience  and 
cheapness. 

Peptone,  —  Witte's  dry  peptone  prepared  from  meat. 

Gelatin, —  best  French  (gold  label)  brand.  It  is  impossible 
to  obtain  gelatin  that  has  a  constant  composition.  It  should 
be  as  free  as  possible  from  acids,  and  a  10  per  cent  solution, 
when  prepared  as  directed,  should  not  soften  at  25°  C. 

Agar,  —  commercial  agar  in  threads  of  as  high  a  grade  as 
can  be  obtained.  Agar  is  prepared  from  certain  seaweeds  of 
Ceylon  and  Japan,  and  is  to  be  found  on  the  market  in  the 
form  of  threads,  square  sticks,  and  as  a  powder. 

Sugars,  —  saccharose,  lactose,  and  anhydrous  dextrose, 
used  for  the  modification  of  standard  media,  should  be  as 
nearly  as  possible  chemically  pure.  The  two  latter  should 
be  used  in  a  powdered  form.  , 

Litmus, —  the  ordinary  litmus  cubes  contain  but  a  small 
amount  of  the  pigment  desired,  azolitmin.  It  is  preferable 
to  purchase  a  purified  form  of  litmus,  which  should  be  free 
from  the  reddish  colorifics  always  found  in  the  cubes,  and 
which  interfere  with  the  sensitiveness  of  the  indicator. 

Reaction  of  media.  The  reaction  of  media  is  expressed 
with  reference  to  the  neutral  point  of  phenolphthalein,  and 


6  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

is  stated  in  terms  of  the  quantity  of  normal  acid  or  alkali 
required  to  neutralize  the  medium.  A  medium  having  an 
acidity  which  would  require  the  addition  of  10  cc.  of  normal 
alkali  to  neutralize  1000  cc.  is  regarded  as  1  per  cent  acid, 
and  this  condition  is  expressed  as  +  1  per  cent.  If  alkaline, 
so  that  5  cc.  of  normal  acid  is  required  to  render  1000  cc. 
neutral  to  phenolphthalein,  the  reaction  is  expressed  as  —  0.5 
per  cent.  In  milk  and  also  in  the  usual  culture  media  the 
neutral  point  of  phenolphthalein  is  not  identical  with  that  of 
litmus,  due  to  the  different  behavior  of  these  indicators  with 
acid  salts.  The  standard  reaction  of  culture  media  for  general 
use  is  +  1  per  cent. 

Neutralization  of  media.  The  following  reagents  will  be 
required:  twentieth  normal  sodium  hydrate  (N/20  NaOH), 
normal  sodium  hydrate  (N/l  NaOH),  normal  hydrochloric 
acid  (N/l  HC1),  and  an  alcoholic  solution  of  phenolphthalein 
(5  grams  of  the  compound  in  1000  cc.  of  50  per  cent  alcohol) 
to  be  used  as  the  indicator.  The  phenolphthalein  should  be 
dissolved  in  the  alcohol  and  dilute  (N/20)  sodium  hydrate 
solution  added  until  a  very  faint  pink  color  is  noted. 

With  a  pipette  place  5  cc.  of  the  medium  to  be  tested  in  a 
small  flask  or  evaporating  dish,  add  45  cc.  of  distilled  water, 
and  boil  one  minute  to  drive  off  C02,  which  affects  the  indi- 
cator. Add  5  drops  of  the  indicator  and  titrate  while  hot  with 
the  N/20  NaOH.  A  faint  but  distinct  pink  color  marks  the 
true  end  point.  Each  0.1  cc.  of  N/20  NaOH  used  for  5  cc. 
of  medium  is  equal  to  0.1  per  cent  acid;  thus  a  medium 
having  an  acidity  of  +  1  per  cent  should  require  1  cc.  of 
N/20  NaOH  for  5  cc.  of  medium.  The  final  reaction  should 
not  differ  from  that  desired  by  more  than  0.2  per  cent,  i.e. 
the  reaction  should  be  between  -f  0.8  and  -f  1.2  per  cent  in 
case  +  1  per  cent  is  desired. 


CULTURAL  TECHNIQUE 


Filtering  of  media.  Bouillon  may  be  filtered  through 
paper.  The  paper  made  by  Schleicher  &  Schtill,  and  known 
as  No.  520  N,  is  well  adapted  for  the  filtering  of  all  kinds  of 
media.  The  paper  can  be  purchased  in  the  form  of 
folded  filters. 

Gelatin  may  be  filtered  by  placing  in  a  large  fun- 
nel a  small  mass  of  copper  turnings  or  clean  ex- 
celsior ;  over  these  a  small  piece  of  absorbent  cotton  ; 
then  a  layer  of  absorbent  cotton  large 
enough  to  reach  to  the  top  of  the 
funnel.    The  layers  of  cotton  should 
be  thin  so  as  not  to  retain  much  of 
the   medium.    Over   the    absorbent 
cotton  place  a  piece  of  wet  cheese 
cloth.    Pour  the  medium  into  the 
funnel  carefully.   In  case  it  has  been 
correctly  prepared,  it  will  filter 
rapidly  and  be  perfectly  clear. 

Agar  may  be  filtered  in  the  same 
manner  as  gelatin. 

The  media  should  be  placed  in 
test  tubes,  7-8  cc.  in  each  tube. 
Extreme  care  should  be  taken  to 
avoid  wetting  the  upper  part  of  the 
tube  with  the  media,  as  this  causes  the  cotton  to  adhere  to 
the  glass.  A  funnel  arranged  as  illustrated  in  Fig.  4  is  con- 
venient; the  glass  tip  should  be  slightly  expanded  at  the 
lower  end,  and  long  enough  so  it  can  be  held  between  the 
fingers  and  thus  kept  out  of  contact  with  the  wall  of  the  tube. 
The  pinchcock  should  be  closed  rapidly  so  that  no  dripping 
will  occur,  and  thus  avoid  soiling  the  mouth  of  the  tube.  The 
storage  flasks  should  not  be  filled  more  than  two  thirds  full ; 


FlG.   3.     Al'l'AUATLS   FOR 
FILTERING   MEDIA 

The  absorbent  cotton  is 
supported  by  excelsior 


FIG.  4.    APPARATUS  FOR  TUBING  MEDIA 

By  holding  the  tip  as  shown,  soiling  the  mouth  of  the  tuhe  with  the 
medium  is  easily  prevented 


CULTURAL  TECHMQUK  9 

if  more  nearly  full,  the  cotton  plug  is  likely  to  be  wet  by  the 
medium  during  sterilization. 

Plugging  tubes.  The  test  tubes  filled  with  media  are  to 
be  plugged  with  cotton  at  once.  Ordinary  roll  cotton  (not 
absorbent)  is  used  for  this  purpose.  A  piece  of  cotton  should 
be  pulled  from  the  roll,  folded  upon  itself  twice,  the  cotton 
compressed  with  the  thumb  and  fingers  and  gradually  forced 
into  the  tube.  The  plug  should  extend  into  the  tube  at  least 
1.5  cm.  and  about  the  same  distance  above  it,  in  order  to 
protect  the  lip  of  the  tube  from  dust  and  to  enable  one  to 
remove  the  plug  easily. 

The  tubes  may  also  be  plugged  rapidly  by  pulling  off  a 
sufficient  amount  of  cotton  with  a  pair  of  dissecting  forceps 
and  thrusting  it  into  the  tube  by  means  of  the  forceps. 

Sterilization  of  media.  Media  in  tubes  or  flasks  is  steril- 
ized by  an  exposure  to  streaming  steam  at  a  temperature  of 
100°  C.  from  fifteen  to  sixty  minutes,  depending  on  the  size 
of  the  containers,  on  each  of  three  consecutive  days,  or  by 
a  single  exposure  to  a  considerably  higher  temperature,  as  in 
steam  under  pressure  in  a  closed  chamber  (autoclave). 

Various  types  of  sterilizers  are  used.  The  Arnold  is  most 
convenient  for  ordinary  laboratory  purposes,  as  it  heats  rapidly 
and  does  not  allow  large  quantities  of  steam  to  escape  into 
the  room.  The  media  in  tubes  should  be  heated  for  twenty 
minutes  on  three  consecutive  days.  With  flaaks  the  period  of 
exposure  should  be1  extended  in  proportion  to  the  size  of  the 
containers.  The  time  should  be  computed  from  the  moment 
when  steam  is  freely  generated  in  the  sterilizer.  During  the 
interval  between  heatings,  the  media  should  be  kept  at  room 
temperature,  in  order  to  allow  the  spores  present  to  germi- 
nate. This  system  of  sterilization  is  known  as  the  intermit- 
tent or  discontinuous  method. 


10 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


In  the  autoclave  a  higher  temperature  is  employed,  and  all 
resistant  forms  are  destroyed  in  one  heating.    In  order  to  be 


FIG.  5.    COTTON  PLUGS 

A,  a  well-made  plug ;  B,  a  plug  that  will  easily  become  displaced ;  C,  a  plug 
that  does  not  protect  the  mouth  of  the  tube  from  dust 

successful  in  the  use  of  the  autoclave  certain  points  must  be 
noted.  Before  using,  care  should  be  taken  to  observe  whether 
there  is  a  sufficient  supply  of  water  in  the  autoclave.  After 


CULTURAL  TECHNIQUE 


11 


GELATIN 
1  PER  CENT  LACTOSE 
+  155        IO/I6/08 
INTERMITTENT. 


closing  the  apparatus  the  vent  must  be  left  open  until  the 
air  is  completely  expelled  by  the  generating  steam,  which 
should  issue  freely.  In  a  mixture  of  air  and  steam  the  tem- 
perature does  not  correspond  to  the  pressure  noted.  Five 
pounds  of  steam  pressure  produces  a  temperature  of  109°  C. ; 
10  pounds,  115°  C.;  15  pounds,  121°  C.  In  all  cases  the 
apparatus  must  be  allowed  to 
cool  off  slowly  and  the  vent 
opened  very  slightly ;  other- 
wise the  pressure  will  de- 
crease more  rapidly  than  the 
temperature  of  the  media. 
This  will  cause  the  media  to 
boil  so  violently  that  the  plugs 
may  be  forced  from  the  con- 
tainers and  a  portion  of  the 
media  lost.  This  is  especially 
true  in  the  case  of  media  in 
flasks.  Media  in  tubes  should 
be  heated  to  120°  C.  (15 
pounds  pressure)  for  five  min- 
utes ;  in  flasks  the  time  must 
be  extended. 

Care  Of  media.    If  possible    FlG-  6-   FORM  OF  LABEL  FOR  MEDIA 

the  media  should  all  be  placed    In  each  basket, of  media  should  be 

;  placed  a  slip  stating  kind  of  medium, 

111  tubes  at  the  time  it  IS  pre-  reaction,  date  of  preparation,  and 
pared,  and  thus  avoid  the  manner  of  sterilization 

changes  due  to  prolonged  and  repeated  heating  in  the  succes- 
sive sterilizations  necessary  when  the  media  is  first  sterilized 
in  bulk  and  again  when  placed  in  tubes.  After  sterilization 
the  media  should  be  kept  at  room  temperature  for  several 
days  and  examined  from  day  to  day.  If  any  "  specks "  in 


12  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

gelatin  or  agar  are  detected,  or  the  least  cloudiness  in  bouillon 
appears,  the  medium  is  not  sterile  and  must  be  immediately 
resterilized.  As  far  as  possible  media  that  are  not  to  be  used 
at  once  should  be  stored  in  an  ice  box  to  prevent  evapora- 
tion. In  case  of  media  that  are  to  be  held  in  storage  for  a 
considerable  period,  or  incubated  for  some  time  at  blood  heat, 
the  containers  may  be  sealed  by  dipping  the  lower  part  of 
the  cotton  plugs  in  melted  paraffin,  or  by  placing  a  cap  of  tin 
foil  over  the  cotton  plug  before  sterilizing. 

In  each  basket  of  culture  medium  should  be  placed  a  slip 
of  paper  stating  the  kind  of  medium,  its  reaction,  the  date  of 
manufacture,  and  manner  of  sterilization.  As  far  as  conven- 
ient, culture  media  should  be  made  in  considerable  quantities, 
so  as  to  increase  uniformity  of  conditions  in  culture  work. 

Preparation  of  broth.  Nutrient  broth  is  prepared  as  fol- 
lows: Infuse  500  grams  (18  ounces)  of  chopped  lean  beef  in 
1000  cc.  of  distilled  water  at  refrigerator  temperature  for  24 
hours.  This  should  be  done  in  a  vessel  of  glass  or  enameled 
iron.  Strain  the  infusion  through  cheese  cloth  or  cotton  flan- 
nel, pressing  out  the  liquid  until  the  meat  appears  dry.  Place 
this  infusion  in  a  weighed  media  cooker,  and  add  water  to 
bring  the  weight  of  the  infusion  to  1000  grams.  Add  1  per 
cent  peptone  (10  grams  per  liter)  and  warm  gently,  not  above 
50°  C.  on  the  water  bath,  until  the  peptone  is  dissolved.  Ti- 
trate two  portions  of  5  cc.  each,  and  bring  the  reaction  to  +  1 
per  cent  by  the  addition  of  N/l  NaOH.  Titrate  another  por- 
tion of  5  cc.  It  will  usually  be  found  that  the  calculated 
amount  of  N/l  NaOH  is  not  sufficient  to  bring  the  reaction 
to  the  desired  point,  due  to  the  fact  that  the  amount  of 
N/20  NaOH  necessary  to  neutralize  5  cc.  of  the  medium 
diluted  with  45  cc.  of  water  does  not  exactly  correspond 
with  the  amount  of  N/l  NaOH  necessary  to  neutralize  the 


CULTURAL  TECHNIQUE  13 

undiluted  medium.  The  reason  for  this  is  not  known.  Hence 
the  titration  must  be  made  after  the  addition  of  the  first  quan- 
tity of  N/lNaOH.  Usually  it  will  be  found  that  a  small 
quantity  of  the  N/l  NaOH  must  be  added. 

After  neutralizing,  cook  thirty  minutes  over  boiling  water ; 
then  boil  for  two  minutes  over  the  free  flame,  stirring  con- 
stantly to  prevent  burning.  Weigh  and  restore  loss  by  evapo- 
ration, allow  the  medium  to  stand  several  minutes  in  order 
that  the  coagulated  proteids  may  settle,  filter  through  paper, 
titrate,  and  record  final  reaction.  If  the  reaction  varies  more 
than  0.2  per  cent  either  way  from  the  normally  accepted 
standard  (+  1  per  cent),  it  must  be  corrected. 

The  finished  product  should  have  a  pale  straw  color,  be 
perfectly  clear,  and  on  boiling  a  sample  in  a  test  tube  should 
show  no  further  precipitation  of  proteid  matter.  Place  in 
flasks  and  tul><-s  and  sterilize  in  the  autoclave. 

Summary : 
Infuse  meat. 

Weigh  cooker  and  record  weight. 
Bring  weight  of  infusion  to  1000  grams. 
Add  1  per  cent  peptone. 

Warm  to  dissolve  peptone,  not  heating  above  50°  C. 
Titrate  and  bring  reaction  to  4- 1  per  cent. 
Cook  thirty  minutes  over  boiling  water. 
Boil  two  minutes  over  free  flame,  stirring  constantly. 
Restore  weight. 
Allow  coagulum  to  settle. 
Filter. 

Test  reaction  and  record. 
Tube  and  sterilize. 

Exercise.    Each  student  will  prepare  1  liter  of  bouillon. 


14  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Preparation  of  gelatin.  Prepare  gelatin  as  follows :  In- 
fuse meat,  strain,  weigh  cooker,  and  bring  weight  of  infusion 
to  1000  grams,  as  in  the  preparation  of  bouillon.  Add  1  per 
cent  of  peptone  and  10  per  cent  of  gelatin.  Warm  on  water 
bath  until  peptone  and  gelatin  are  dissolved,  not  allowing 
the  temperature  to  rise  above  50°  C.  Titrate  and  bring  re- 
action to  -}- 1  per  cent.  Eestore  weight,  taking  into  account 
the  weight  of  the  gelatin  added.  Cook  thirty  minutes  over 
boiling  water.  Boil  two  minutes  over  free  flame,  stirring 
constantly. 

Allow  liquid  to  stand  a  few  moments  to  settle.  Filter 
through  cotton  (see  p.  7).  Test  reaction,  adjust  if  necessary. 
Eecord  final  reaction.  Tube  and  sterilize.  The  filtering  of 
gelatin  is  more  difficult  than  in  the  case  of  broth.  No  trouble 
will  be  experienced  if  the  filtering  is  not  attempted  until  the 
coagulum  is  firm  and  solid  and  the  liquid  gelatin  appears 
perfectly  clear.  If  the  proteids  are  not  firmly  coagulated, 
gelatin  is  very  difficult  to  filter,  as  the  fine  sediment  clogs 
the  pores  of  the  filter.  If  the  cooking  is  continued  until  the 
albumen  is  completely  coagulated  and  the  funnel  is  arranged, 
as  shown  in  Fig.  3,  no  trouble  should  be  experienced. 

Prolonged  heating  injures  the  solidifying  properties  of 
gelatin.  If  the  sterilizing  is  done  by  the  discontinuous 
method,  the  gelatin  should  be  placed  in  cold  water  after 
each  heating.  If  the  autoclave  is  employed,  the  exposure 
should  not  exceed  five  minutes  at  120°  C.  (15  pounds  pres- 
sure). After  removal  from  the  autoclave  the  gelatin  should 
be  cooled  at  once  by  placing  in  cold  water.  It  is  preferable 
to  use  but  7-10  pounds  pressure  and  continue  the  exposure 
for  ten  or  twelve  minutes. 

Exercise.    Each  student  will  prepare  1  liter  of  gelatin. 


CULTURAL  TECHNIQUE  15 

Preparation  of  agar.  Infuse  500  grains  of  meat  in  500  cc. 
of  distilk'tl  \vater.  Strain  the  infusion.  Weigh  the  vessel,  and 
bring  weight  of  infusion  to  500  grams.  Add  10  grams  of  pep- 
tone. Warm  over  a  water  bath  until  peptone  is  dissolved. 
Heat  500  cc.  of  water  in  a  weighed  vessel  until  it  is  boiling 
rapidly.  Add  15  grams  of  thread  agar  to  the  boiling  water, 
continue  the  heating  with  constant  stirring  until  the  agar  is 
completely  dissolved,  restore  loss,  and  allow  the  whole  to  cool 
to  55°  C.  The  agar  may  also  be  dissolved  by  heating  hi  the 
autoclave  for  ten  or  fifteen  minutes  at  120°  C.  To  the  500 
grams  of  meat  infusion,  which  should  have  a  temperature  of 
45-50°  C.,  add  the  agar  solution,  keeping  the  temperature 
below  60°  C.  Bring  weight  of  mixture  to  10QO  grams.  Titrate 
and  bring  reaction  to  +  1  per  cent.  Heat  thirty  minutes  over 
boiling  water.  Boil  two  minutes  over  a  free  flame,  stirring 
constantly,  as  the  agar  is  very  likely  to  burn.  Eestore  weight. 
Filter  as  in  the  case  of  gelatin.  Test  reaction,  and  adjust  if 
necessary.  Eecord  final  reaction.  Tube  and  sterilize. 

The  filtering  of  agar  is  likely  to  be  difficult  unless  the 
medium  has  been  correctly  prepared.  The  points  to  be  noted 
are :  (1)  to  obtain  a  perfect  solution  of  the  agar  by  prolonged 
boiling,  or  heating  in  the  autoclave;  (2)  to  produce  a  firm 
coagulation  of  the  proteid  matter  by  subsequent  heating  until 
the  mass  of  the  medium  appears  clear.  The  removal  of  the 
fine  turbidity  in  agar  can  only  be  accomplished  through  its 
collection  by  the  coagulating  proteid,  which  is  readily  re- 
moved by  filtration.  If  an  attempt  is  made  to  filter  before 
the  medium  is  in  proper  condition,  it  will  result  in  loss  of 
time  and  material.  If  a  heavy  granular  coagulum  does  not 
form  upon  prolonged  heating,  allow  the  medium  to  stand 
until  the  next  day,  break  up  the  mass  with  a  stirring  rod, 
and  melt  over  boiling  water.  This  will  usually  result  in  a 


16  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

more  complete  flocculation  of  the  proteid  so  as  to  permit 
ready  filtration. 

Agar  is  not  injured  by  prolonged  heating,  as  is  gelatin.  The 
finished  medium  should  he  clear  when  liquid,  and  slightly 
opaque  when  solid.  It  should  be  of  a  firm  jelly-like  consist- 
ency, not  soft,  mushy,  or  easily  broken. 

Exercise.    Each  student  will  prepare  1  liter  of  agar. 

It  frequently  happens  in  the  preparation  of  media  that  the 
material  becomes  seeded  with  resistant  spore-bearing  organ- 
isms. These  do  not  come  from  the  materials  used  in  the 
media,  but  frequently  from  unclean  utensils.  All  utensils 
—  media  cookers,  funnels,  pipettes,  etc.  —  should  therefore 
be  washed  with  hot1  water  and  drained  as  soon  as  possible,  in 
order  to  free  them  from  the  adhering  media.  If  allowed  to 
stand,  germ  growth  takes  place  in  this  material,  and  the 
utensils  become  so  abundantly  seeded  as  to  make  sterili- 
zation of  the  media  prepared  in  such  vessels  difficult  or 
impossible. 

Meat  infusion  should  be  used  whenever  possible  for  media, 
but  beef  extract  can  be  substituted,  3  grams  of  Liebig's  Beef 
Extract  being  used  for  each  liter  of  medium.  Egg  albumen 
must  then  be  used  as  a  clearing  agent,  to  remove  the  finely 
dividend  material.  The  white  of  one  egg  for  each  liter  of  me- 
dium should  be  added  to  the  distilled  water  before  adding 
the  other  ingredients.  The  albumen  should  be  well  beaten 
and  thoroughly  mixed  with  the  water.  Then  proceed  as  when 
meat  infusion  is  used. 

Preparation  of  milk.  The  milk  to  be  used  as  a  culture 
medium  should  be  as  fresh  as  possible.  It  should  not  have 
an  acidity  above  0.18  per  cent  calculated  as  lactic  acid.  The 
normal  acidity  of  perfectly  fresh  milk  ranges  from  0.12  to 


CULTURAL  TECHNIQUE  17 

0.18  per  cent  of  lactic  acid,  equaling  +  1.3  to  +  1.6  per  cent 
on  the  arbitrary  scale  ordinarily  employed  for  media. 

On  heating,  the  acidity  is  reduced  by  the  precipitation  of 
the  calcium  salts  and  the  expulsion  of  the  C02,  so  that  a 
milk  having  an  acidity  of  0.18  per  cent  calculated  as  lactic 
acid  will  show  not  more  than  +  1.2  per  cent  when  tested 
after  sterilization  by  the  usual  method.  It  is  possible  to  neu- 
tralize milk  showing  a  high  degree  of  acidity,  but  it  is  not 
advisable,  since  within  certain  limits  the  acidity  is  a  measure 
of  purity,  and  even  under  the  best  of  conditions  milk  is  very 
difficult  to  sterilize.  The  whole  milk  may  be  allowed  to  cream 
in  a  refrigerator  by  gravity,  and  the  skim  milk  siphoned  off, 
or,  preferably,  the  milk  may  be  skimmed  in  a  centrifugal 
machine  or  cream  separator. 

Milk  is  used  either  as  plain  milk  or  in  the  form  of  litmus 
milk  prepared  by  the  addition  of  a  litmus  solution.  The 
litmus  solution  should  be  added  to  the  milk  before  tubing, 
and  in  sufficient  amount  to  give  it  a  decidedly  blue  color. 
Milk  is  preferably  sterilized  by  the  discontinuous  method. 
Litmus  is  easily  reduced  at  100°  C.  by  the  action  of  the  milk 
sugar.  Thus  when  litmus  milk  is  removed  from  the  sterilizer, 
it  may  appear  almost  colorless.  The  color  soon  reappears  on 
exposure  to  the  air,  due  to  the  oxidation  of  the  reduced  in- 
dicator. The  sterility  of  the  milk  must  be  tested  by  allowing 
it  to  stand  a  week,  if  possible,  before  using* 

Preparation  of  sugar  media.  Gelatin,  agar,  and  bouillon 
are  often  amended  by  the  addition  of  sugars  (saccharose,  dex- 
trose, and  lactose).  Sugar  media  should  be  sterilized  by  the 
discontinuous  method,  and  the  reaction  should  be  neutral  to 
phenolphthalein.  Saccharose  is,  however,  the  only  one  of  the 
three  sugars  easily  affected,  being  changed  in  part  (inverted)  to 
dextrose  and  levulose  when  heated  in  acid  solutions.  Lactose 


18  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

is  not  so  easily  decomposed,  and  media  containing  it  may  be 
sterilized  in  the  autoclave  even  when  acid.  When  it  is  desir- 
able to  test  the  properties  of  an  organism  with  reference  to 
its  conduct  towards  certain  sugars,  care  should  be  taken  to 
have  no  other  sugar  present  in  the  medium  than  the  one 
added,  and  to  have  this  present  in  an  unchanged  form. 

Media  prepared  from  meat  infusion  will  contain  some 
muscle  sugar  (inosite).  This  may  be  removed  from  the  in- 
fusion by  seeding  the  same  with  an  organism  able  to  fer- 
ment this  sugar  (B.  coli  communis),  incubating  from  sixteen 
to  twenty  hours  at  37°  C.  and  then  proceeding  as  usual  in 
the  preparation  of  the  medium. 

Preparation  of  litmus  solution.  The  litmus  ordinarily 
purchased  varies  so  widely  in  its  content  of  the  pigment 
desired,  azolitmin,  that  it  is  impossible  to  give  definite 
directions  for  its  preparation.  Ten  grams  of  Kahlbaum's  or 
Schuchardt's  litmus  powder  are  extracted  with  500  cc.  of 
water  by  placing  over  night  in  a  warm  place ;  then  filter  and 
tube.  Sterilize  by  the  discontinuous  method. 

Preparation  of  water  blanks.  For  dilution  purposes  in 
quantitative  work  sterile  water  must  be  employed.  It  is  cus- 
tomary to  use  the  so-called  physiological  salt  solution,  pre- 
pared by  the  addition  of  0.6  per  cent  of  common  dairy  salt 
to  distilled  water,  in  place  of  the  distilled  water  which  may 
exert  an  injurious  effect  on  the  bacteria  by  reason  of  its 
osmotic  properties. 

Various  modifications  of  the  above  media  are  used  in  bac- 
teriological work,  as  well  as  many  other  types  of  media,  such 
as  potato,  blood  serum,  egg,  etc.  These,  however,  are  not  usu- 
ally employed  in  ordinary  dairy  work,  and  for  their  prepara- 
tion and  use  the  student  is  referred  to  books  on  general  and 
medical  bacteriology. 


CULTURAL   TECHNIQUE  19 

Quantitative  analysis  of  milk.  The  number  of  bacteria 
present  in  any  material  is  determined  by  making  "  plate  cul- 
tures "  from  it.  A  definite  quantity  is  placed  in  a  Petri  dish, 
and  a  tube  of  one  of  the  liquefiable  solid  media  is  poured  into 
the  Petri  dish  and  intimately  mixed  with  the  milk.  The 
liquid  medium  spreads  in  a  thin  layer  over  the  bottom  of 
the  plate,  which  is  placed  on  a  level  surface  to  cool,  thus 
allowing  the  medium  to  solidify.  The  bacterial  cells  dis- 
tributed in  the  liquid  medium  are  held  in  place  when  the 
medium  solidifies.  The  resulting  growth  thus  appears  as  dis- 
tinct spots,  called  "  colonies,"  each  of  which  is  the  progeny 
of  a  single  type  of  organism,  although  not  necessarily  of  a 
single  cell.  The  counting  of  these  colonies  after  a  sufficient 
time  has  elapsed  for  their  growth  will  give  the  approximate 
number  of  living  bacterial  cells  present  in  the  original  sub- 
stance at  the  time  the  plate  cultures  were  made.  This  cul- 
ture method,  originally  devised  by  Koch,  has  been  of  greatest 
value  in  placing  bacteriological  study  on  a  strictly  scientific 
foundation. 

It  is  impossible  by  any  method  yet  devised  to  determine 
the  absolute  number  of  bacteria  present  in  any  substance, 
which,  like  milk,  contains  under  natural  conditions  a  variety 
of  forms  differing  in  requirements  for  their  growth.  The  col- 
onies that  appear  on  the  plate  cultures  represent  the  organisms 
that,  under  the  given  conditions  of  food,  temperature,  and  air 
supply,  find  favorable  conditions  for  development.  If  another 
food  substance  is  used,  if  the  reaction  of  the  medium  is  varied 
widely,  or  if  the  temperature  is  changed,  a  different  class  of 
bacteria  will  be  found  on  the  plate  cultures.  Hence  the  plate 
method  of  determining  the  number  of  bacteria  in  milk  or  any 
substance  is  not  an  absolute  one,  and  one  which  gives  com- 
parable results  only  under  constant  conditions. 


20  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Each  colony  represents  a  living  bacterial  cell  in  the  sub- 
stance plated,  only  when  the  cells  are  uniformly  distributed 
throughout  the  liquid  and  no  clumps  of  cells  persist.  This 
condition  is  impossible  to  attain  in  viscous  liquids  and  in 
those  in  which  growth  takes  place ;  hence,  on  culture  plates, 
many  colonies  represent  the  progeny  of  a  clump  of  cells, 
numbering  many  score  or  more,  instead  of  being  the  result 
of  the  growth  of  a  single  cell.  The  results  obtained  from  the 
examination  of  any  sample  will  thus  vary  with  the  care  used 
in  obtaining  an  even  distribution  of  the  bacteria.  If  a  sample 
of  milk  is  allowed  to  stand  for  twenty-four  hours  and  plate 
cultures  prepared  before  it  has  been  shaken,  and  another  set 
after  the  -milk  has  been  shaken  for  several  minutes,  the  re- 
sults will  vary  widely,  due  to  the  more  or  less  complete  rup- 
ture of  the  cell  clumps  of  the  bacteria. 

If  the  colonies  on  the  plates  are  very  numerous,  they  will 
consist  mainly  of  those  kinds  that,  under  a  given  environment, 
find  most  favorable  conditions  for  growth.  Those  finding  less 
favorable  conditions  are  inhibited.  If  lactose-agar  plates  are 
seeded  with  a  sample  of  sour  milk  and  kept  at  25°- 30°  C., 
the  ordinary  sour  milk  or  lactic-acid  bacteria  will  grow  very 
rapidly,  and  the  putrefactive  bacteria  will  not  develop  ;  but  if 
the  cultures  are  made  with  gelatin  containing  no  milk  sugar, 
the  sour  milk  bacteria  will  develop  very  sparsely,  or  not  at 
all,  and  only  the  other  type,s  will  be  found  on  the  plates. 

If  the  colonies  are  less  numerous,  so  that  each  is  not  within 
the  zone  of  influence  of  other  developing  colonies,  this  in- 
hibition will  not  take  place.  Generally  such  inhibition  or 
"antagonism"  is  caused  by  the  effect  of  the  soluble  by- 
products of  one  form  diffusing  throughout  the  medium. 

For  ideal  results  each  colony  should  develop  as  though  it 
were  the  only  colony  on  the  plate.  This  condition  is  of  course 


CULTURAL  TECHNIQUE  21 

difficult  to  obtain.  The  extent  of  the  zone  of  influence  of  a 
colony  depends  upon  the  organism  and  the  medium.  Inhibi- 
tion by  overcrowding  is  negligible  when  the  number  of  col- 
onies ranges  from  100  to  200  on  a  90-mm.  plate. 

If  larger  numbers  are  present,  this  inhibitory  action  becomes 
pronounced  and  a  reduction  in  total  content  is  to  be  observed. 
This  error,  as  well  as  tedious  enumeration  of  very  small  col- 
onies, makes  it  preferable  to  have  properly  prepared  plate  cul- 
tures. In  plates  prepared  from  pure  cultures  of  bacteria,  i.e. 
organisms  all  of  one  kind,  this  inhibiting  tendency  is  much 
less  important. 

Dilution.  The  number  of  bacteria  present  in  any  given 
material,  as,  for  instance,  milk,  is  of  course  unknown.  Hence, 
in  preparing  plate  cultures,  in  order  to  have  the  proper  num- 
ber of  colonies  it  is  necessary  to  vary  considerably  the  quan- 
tities of  milk  used  in  the  plates.  The  milk  should  be  diluted 
with  sterile  water  or  salt  solution  (water  blanks).  The  extent 
to  which  the  dilution  must  be  carried  cannot  be  determined 
with  exactness,  but  with  experience  the  analyst  can  judge 
from  the  age  of  the  milk,  the  conditions  under  which  it  was 
produced,  the  temperature  at  which  it  has  been  kept,  and,  by 
determination  of  the  acidity,  the  dilutions  which  are  most 
likely  to  give  satisfactory  results. 

In  the  case  of  perfectly  fresh  milk,  plates  should  be  made 
with  1/10,  1/100,  and  1/1000  cc.;  ordinary  market  milk 
1/100,  1/1000,  1/10,000  cc.;  milk  having  an  acidity  over 
0.2  per  cent,  1/1000,  1/10,000,  and  1/100,000  cc.  In  the 
case  of  sour  milk  and  cream  it  will  be  necessary  to  use  still 
greater  dilutions.  , 

The  various  dilutions  are  obtained  by  the  use  of  9-cc.  and 
99-cc.  water  blanks  in  test  tubes  and  150-cc.  Erlenmeyer 
flasks.  To  obtain  0.1  cc.  of  milk,  1  cc.  is  placed  in  a  9-cc. 


22  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

water  blank  by  means  of  a  sterile  1-cc.  pipette,  the  liquid  well 
mixed,  and  1  cc.  of  the  mixture  taken.  To  obtain  1/100  cc. 
of  milk,  1  cc.  is  placed  in  99  cc.  of  water  and  1  cc.  taken ; 
1/1000  cc.  of  milk  is  obtained  by  carrying  1/100  cc.  to  a  9-cc. 
water  blank  and  using  1  cc.  of  the  mixture.  Still  higher  dilu- 
tions are  obtained  in  a  similar  manner.  In  each  plate  is  placed 
the  same  amount  of  the  mixture  of  milk  and  water,  —  1  cc, ; 
thus  the  extent  to  which  the  medium  is  diluted  is  constant. 
A  clean  pipette  must  be  employed  for  each  dilution. 

Media.  A  variety  of  media  are  used  for  the  quantitative 
analysis  of  milk.  Each  has  certain  advantages  and  disadvan- 
tages. Gelatin  melts  at  temperatures  above  25°  C.,  and  is 
liquefied  by  certain  kinds  of  bacteria.  If  these  forms  are 
numerous,  the  entire  mass  of  gelatin  will  be  rapidly  lique- 
fied and  no  definite  results  obtained.  Agar  melts  at  96°- 
99°  C.  and  does  not  solidify  again  until  it  has  cooled  to 
39°-40°  C.  It  is  not  liquefied  by  any  of  the  ordinary  bac- 
teria. Gelatin  can  therefore  be  used  only  at  room  tempera- 
ture, while  agar  cultures  may  be  incubated  at  blood  heat. 
Lactose  agar  is  preferable  for  quantitative  analysis  of  milk. 

Cooling.  After  the  liquid  medium  has  been  poured  into  the 
plates  they  must  be  tilted  from  side  to  side  in  order  to  dis- 
tribute the  medium  evenly  over  the  entire  inner  floor  of  the 
dish.  This  must  be  done  before  the  medium  begins  to  solidify, 
so  that  the  appearance  of  the  plate  shall  not  be  marred  by  a 
rough  surface.  This  is  easily  accomplished  with  gelatin,  as 
it  cools  slowly.  With  agar  the  medium  must  be  allowed 
to  cool  to  43°-45°  C.  before  it  is  inoculated,  and  since  it 
solidifies  at  39°-40°  C.,  it  requires  more  careful  manipula- 
tion to  properly  prepare  cultures  with  this  medium.  More 
uniform  distribution  can  be  had  with  agar  if  before  adding 
the  medium  the  plates  are  slightly  warmed  by  holding  them 


CULTURAL  TECHNIQUE  23 

in  the  hand  and  passing  them  quickly  through  the  flame  of 
a  Bunseii  burner.  After  the  culture  medium  is  distributed 
the  plates  should  be  placed  on  a  perfectly  level  surface  to 
solidify,  in  order  that  the  culture  medium  may  be  uniform 
in  thickness  and  the  colonies  evenly  distributed. 

Incubation.  Nearly  all  forms  of  bacteria  found  in  milk 
grow  at  room  temperature,  about  20°  C.,  although  higher 
temperatures  hasten  the  growth  of  many  kinds. 

Two  temperatures  are  ordinarily  used  in  bacteriological 
laboratories, —  20°  C.  (room  temperature)  and  37.5°  C.  (blood 
heat).  In  order  to  maintain  a  constant  temperature  an  ap- 
paratus known  as  a  thermostat  or  incubator  is  used.  A 
temperature  of  37.5°  C.  is  maintained  by  a  gas  flame,  the 
gas  supply  being  regulated  by  a  thermoregulator.  A  constant 
temperature  of  20°  C.  is  difficult  to  maintain,  since  under 
summer  conditions  room  temperature  often  exceeds  this 
limit.  If  an  abundant  supply  of  cold  water  is  available,  the 
thermostat  may  be  kept  cool,  i.e.  at  a  temperature  below 
20°  C.,  and  then  heated  with  a  small,  regulated  gas  flame. 

Gelatin  cultures  must  always  be  incubated  at  tempera- 
tures below  25°  C.  Agar  cultures  may  be  kept  at  any 
desired  temperature  up  to  the  maximum  for  bacterial  growth, 
which  for  most  species  does  not  exceed  40°-45°  C. 

The  proper  time  of  incubation,  i.e.  the  period  between  the 
preparation  and  the  counting  of  the  plates,  \vill  depend  upon 
the  temperature.  If  37.5°  C.  is  used,  the  number  of  colonies 
will  not  materially  increase  after  forty-eight  hours  incuba- 
tion. The  individual  colonies  will,  however,  increase  in  size 
and  make  the  counting  easier.  This  is  especially  true  if  the 
plates  are  incubated  for  another  forty-eight  hours  after  they 
are  removed  from  the  37°  C.  incubator.  Plates  kept  at  20°  C. 
should  not  be  counted  before  three  days,  and  the  maximum 


24  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

number  of  colonies  will  not  appear  in  less  than  six  or  eight 
days.  It  is,  however,  often  advisable  to  secure  results  before 
the  expiration  of  such  a  long  period  of  incubation,  and  for 
comparative  purposes  a  shorter  time  than  the  maximum  here 
given  is  frequently  used  as  a  basis  for  official  counts. 

Agar  on  solidifying  expresses  a  slight  amount  of  water. 
This  often  collects  on  the  surface  of  the  medium  in  a  thin 
film,  allowing  the  formation  of  a  spreading  surface  growth. 
Frequently  a  thin,  nearly  transparent  growth  may  spread 
between  the  under  surface  of  the  culture  medium  and  the 
glass.  If  freshly  prepared  plates  are  placed  immediately  in  a 
37°  C.  incubator,  evaporation  from  the  culture  medium  occurs, 
which  permits  of  condensation  on  the  cover  of  the  dish,  and 
the  moisture  sometimes  collects  to  such  an  extent  that  drop- 
lets of  water  fall  on  to  the  medium,  causing  the  formation 
of  a  spreading  growth  and  seriously  interfering  with  subse- 
quent counting.  To  obviate  this,  the  plates  may  be  inverted 
in  the  37°  C.  incubator.  Covers  made  of  porous  earthenware 
are  also  useful  in  preventing  this  trouble. 

Counting.  To  enumerate  the  developing  colonies,  the  cover 
is  removed  from  the  culture  dish  and  the  plate  placed  on 
a  black  surface,  which  is  divided  into  appropriate  divisions, 
in  order  to  facilitate  counting  (Frost's  modification  of  the 
Jeffer's  plate  counter  is  most  satisfactory).  The  counting 
should  be  done  by  the  aid  of  a  hand  lens,  magnifying  5-8 
diameters.  When  possible,  the  entire  plate  should  be  counted. 
Where  the  colonies  are  too  numerous,  an  estimate  may  be 
made  by  counting  a  representative  portion  of  the  plate,  and 
from  the  data  obtained  calculating  the  total  number  of  colo- 
nies on  the  plate.  This  process  is,  however,  often  unsatisfac- 
tory, especially  if  the  colonies  are  not  uniformly  distributed. 
If  the  plate  was  not  allowed  to  solidify  in  a  level  position, 


CULTURAL  TECHNIQUE  25 

the  colonies  will  be  abundant  on  one  side  and  sparse  or 
absent  on  the  other,  making  it  difficult  to  secure  a  correct 
approximation.  In  case  the  count  to  be  made  is  not  the 
final  one,  or  if  the  culture  is  to  be  used  for  other  purposes, 
the  cover  of  the  dish  must  not  be  removed  during  the 
counting  process. 

A  number  of  plate  cultures  must  be  made  from  each 
sample  of  milk.  Not  all  of  these  are  to  be  counted,  but 
only  those  having  the  most  desirable  number  of  colonies.  If 
plates  have  been  made  with  1/100,  1/1000,  and  1/10,000  cc. 
of  milk,  the  colonies  on  the  first  plate  of  the  series  may  be 
so  numerous  as  to  render  counting  difficult  or  impossible. 
The  next  plate,  containing  only  1/10  as  much,  may  have 
several  hundred,  while  the  final  one  of  the  series,  made  with 
1/10,000  cc.,  should  have  only  a  few  score  of  colonies.  To  give 
reliable  and  consistent  results  plate  cultures  should  show  be- 
tween forty  and  two  hundred  colonies.  When  the  number  of 
colonies  is  much  in  excess  of  two  hundred,  inhibition  of  one 
form  by  another  takes  place,  and  increases  with  an  increas- 
ing number  of  colonies.  The  maximum  number  of  colonies 
that  will  develop  on  a  plate  ranges  from  ten  to  fifteen  thou- 
sand. If  the  material  used  in  preparing  the  plate  culture 
contained  a  greater  number  of  organisms,  the  results  will  not 
be  accurate.  If  the  colonies  are  few  in  number  on  the  plate, 
it  is  difficult  to  obtain  concordant  results.  For  these  reasons 
plates  which  do  not  approximate  the  number  of  colonies 
considered  as  desirable  should  be  discarded.  The  average  of 
all  plates  counted  should  be  taken  and  the  results  expressed 
as  the  number  of  bacteria  per  cubic  centimeter  in  the  milk 
examined. 

Expression  of  results.  Since  it  is  impossible  to  determine 
with  absolute  accuracy  the  number  of  bacteria  present  in  any 


26  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

substance,  fictitious  accuracy  should  be  avoided  in  the  expres- 
sion of  the  results.  In  reporting  the  results  of  the  examina- 
tion of  ordinary  market  milks,  the  Committee  on  Standard 
Methods  of  Milk  Analysis  of  the  American  Public  Health 
Association  has  made  the  following  recommendation : 

From  50,000  to  500,000  bacteria  per  cubic  centimeter 
report  results  to  nearest  50,000. 

From  500,000  to  1,000,000,  to  nearest  100,000. 

From  1,000,000  to  2,000,000,  to  nearest  200,000. 

From  2,000,000  to  5,000,000,  to  nearest  500,000. 

Above  5,000,000,  to  nearest  1,000,000. 

The  error  with  present  methods,  even  with  very  careful 
work,  amounts  to  10-15  per  cent,  so  it  is  evident  that  the 
above  scheme  is  sufficiently  accurate. 

In  reporting  the  results  of  very  clean  milk  (certified  and 
sanitary  milk)  the  exact  figures  should  be  given,  neglecting 
the  first  significant  figure,  as  510  bacteria  per  cubic  centi- 
meter instead  of  514. 

Recapitulation  of  preparation  of  plate  cultures.  The  pro- 
cedure for  the  examination  of  a  sample  of  milk  from  which 
plates  are  to  be  made  containing  1/100, 1/1000,  and  1/10,000 
cc.  of  milk  is  as  follows.  Shake  sample  twenty-five  times. 

Mark  two  99-cc.  water  blanks  1  and  3  respectively,  and 
mark  one  9-cc.  water  blank  2. 

With  a  sterile  pipette  transfer  to  blank  No.  1,  1  cc.  of  milk, 
and  return  pipette  to  milk.  Replace  the  cotton  plug  in  the 
water  flask  and  shake  well.  No  harm  will  be  done  if  the 
cotton  becomes  wet. 

With  another  sterile  pipette,  transfer  1  cc.  from  flask  No.  1 
to  No.  2,  and  also  1  cc.  from  No.  1  to  No.  3.  Return  pipette 
to  No.  1.  The  cotton  plug  need  not  be  replaced.  Mix  Nos.  2 
and  3,  and  in  each  place  a  sterile  pipette. 


CULTURAL  TECHNIQUE  27 

Label  six  sterile  culture  dishes  with  the  laboratory  num- 
ber of  the  sample  of  milk,  date,  and  quantity  of  milk  to  be 
placed  iii  each.  Transfer  to  each  of  two  Petri  dishes  1  cc.  of 
the  first  dilution  (1/100  cc.  of  the  original  milk),  to  two  more 
dishes  1  cc.  of  the  second  dilution  (1/1000  cc.),  and  to  the  two 
remaining  dishes  1  cc.  of  the  third  dilution  (1/10,000  cc.).  ' 

In  placing  the  diluted  milk  in  the  dish  lift  the  cover  at 
one  side,  place  the  tip  of  the  pipette  on  the  bottom  of  the 
dish  and  allow  the  desired  quantity  to  run  out. 

Do  not  remove  the  cover  completely. 

Do  not  allow  the  pipette  to  touch  the  desk  or  any  other 
unsterile  object. 

In  a  metal  cup  partly  filled  with  water  place  seven  tubes 
of  lactose  agar.  Heat  over  a  Bunsen  burner  until  the  agar  is 
completely  melted. 

Place  a  thermometer  in  the  cup,  cool  to  45°  C.  by  adding 
cold  water,  and  allow  the  tubes  to  stand  five  minutes  at  this 
temperature. 

Remove  a  tube,  wipe  off  the  water,  remove  the  cotton  plug. 
Flame  the  open  end  of  the  tube  in  a  Bunsen  flame. 

Lift  one  side  of  the  cover  of  the  culture  dish,  pour  in  the 
culture  medium,  replace  the  cover,  mix  the  medium  with  the 
diluted  milk  by  tipping  the  Petri  dish  from  side  to  side,  and 
place  the  dish  on  a  level  surface. 

Incubate  at  37°  C.  for  forty-eight  hours,  s 

Count  cultures  and  record  results. 

Results  obtained  from  duplicate  plates  should  check  within 
15  per  cent. 

Pour  the  seventh  tube  of  melted  agar  into  an  empty,  sterile 
culture  dish  as  a  check.  Incubate.  If  due  care  has  been  ex- 
ercised and  medium  and  glassware  are  sterile,  no  colonies 
should  develop  in  this  culture. 


28 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


Exercise.  Each  student  will  make  a  quantitative  examination  of 
three  samples  of  milk  :  (1)  fresh  milk;  (2)  ordinary  market  milk  ; 
(3)  milk  having  a  high  acidity  (0.3-0.4  per  cent). 

The  dilutions  to  be  used  in  the  examination  of  the  different  milks 
should  be  indicated  by  the  instructor. 

Arrangement  of  data.  The  complete  data  should  be  ar- 
ranged in  tabular  form  as  far  as  possible,  so  as  to  facilitate 
the  examination  of  the  results.  The  following  may  serve  as 
an  example  of  arrangement : 


LABORATORY 
NUMBER  OF 
SAMPLE 

AMOUNT  OF 
MILK 

COLONIES 

AT  48 

HOURS 

COLONIES 

AT  96 

HOURS 

NUMBER 
PER  CUBIC 
CENTIMETER 

NUM- 
BER OF 
KINDS 

I 

1/100  cc. 

Not  counted 

1/1000  cc. 

Not  counted 

l/10,000cc. 

200 

208 

2,000,000 

4 

II 

1/100  cc. 

Not  counted 

1/1000  cc. 

110 

121 

110,000 

1/10,000  cc. 

14 

15 

140,000 

.5 

Method  for  comparative  results.  When  it  is  desirable  to 
determine  merely  the  relative  number  of  bacteria  in  a  num- 
ber of  samples,  as,  for  example,  in  sweet  and  ripened  cream, 
a  different  method  may  be  used,  which  is  sufficiently  accurate 
for  the  purpose. 

A  standard-sized  loop  is  made  of  No.  27  platinum  wire  by 
making  the  loop  around  an  eight-penny  common  nail  (10| 
wire  gauge).  The  loop  should  be  made  as  illustrated  (Fig.  7).  If 
it  is  always  used  in  the  same  way,  i.e.  immersed  to  the  same 
depth  in  the  liquid,  removed  quickly,  and  the  lumen  of  the 
loop  kept  parallel  to  the  surface  of  the  liquid,  a  double  con- 
vex drop  of  liquid  will  be  removed,  which  will  contain  an  ap- 
proximately uniform  quantity  of  milk  (0.008  to  0.012  grams). 
High  dilutions  can  thus  be  obtained  by  the  use  of  small  water 
blanks  (5  cc.),  and  the  use  of  many  pipettes  avoided. 


CULTURAL  TECHNIQUE  29 

Qualitative  analysis  of  milk.  It  is  frequently  desirable 
to  determine  the  number  of  different  kinds  of  bacteria  in  a 
sample  of  milk,  as,  for  instance,*  acid-producing  organisms, 
organisms  liquefying  or  not  liquefying  the  gelatin.  For  these 
purposes  media  must  be  used  which  will  permit  of  the  de- 
velopment of  the  desired  species. 

In  order  to  determine  the  number  of  acid-producing  and 
liquefying  organisms,  litmus  lactose  gelatin  may  be  used,  pro- 
ceeding exactly  as  in  the  case  of  the  quantitative  examination 
of  milk,  except  that  to  each  tube  of  lactose  gelatin,  after  melt- 
ing, is  added  with  a  sterile  pipette  sufficient  sterile  litmus 
solution  to  give  the  medium  a  decidedly  blue  color.  The  acid- 
producing  bacteria  are  easily  differentiated  on  this  medium 
by  their  red  color  unless  the  plates  are  so  thickly  seeded  with 
acid  colonies  that  the  entire  mass  of  the  medium  is  changed. 
For  qualitative  analysis  thickly  seeded  plates  are  of  little  value. 

This  medium  also  divides  the  bacteria  which  develop  into 
two  classes,  namely:  (1)  liquefiers, — those  which  develop  fer- 
ments capable  of  permanently  rendering  the  gelatin  medium 
liquid;  (2)  those  unable  to  produce  this  change.  The  lique- 
fiers are  further  differentiated  into  those  which  perform  this 
change  very  rapidly  and  those  which  produce  a  restricted 
and  often  deepened  pit  of  liquefied  material  closely  surround- 
ing the  colony. 

The  differentiation  of  the  bacteria  can  be  carried  further 
by  a  study  of  individual  colonies.  These  vaiy  in  size  and 
appearance  with  different  organisms,  and  the  differences  are 
more  or  less  constant.  Especial  use  is  made  of  these  colony 
characteristics  in  the  detailed  study  and  identification  of  the 
various  kinds  of  organisms. 

Exercise.  Prepare  litrnus-lactose-gelatin  plates  from  the  samples 
of  milk  furnished.  Incubate  at  20°  C.  Count  the  number  of  acid 


30     EXPERIMENTAL  DAIRY  BACTERIOLOGY 

colonies,  the  number  of  liquefying  and  nonliquefying  colonies  on 
the  gelatin  plates;  and  express  the  results  as  the  number  of  each 
contained  in  a  cubic  centimeter  of  the  milk. 

Isolation  of  pure  cultures.  A  pure  culture  is  one  which 
contains  but  a  single  kind  of  organism.  Under  natural  con- 
ditions the  bacterial  flora  of  any  substance  is  made  up  of  a 
number  of  kinds.  If  it  is  desired  to  study  the  different  kinds 
in  detail,  a  separation  must  be  made  and  each  obtained  in 
"  pure  culture."  Each  colony  on  the  culture  plate  represents 
the  progeny  of  a  single  cell  or  of  a  number  of  adherent  cells 
of  one  kind;  hence  the  cultures  obtained  from  a  single  col- 
ony are  termed  "pure  cultures."  If  from  the  isolated  colony 
further  cultures  are  made  in  proper  media,  the  morphological 
and  physiological  study  of  the  organism  can  be  carried  out. 


FIG.  7.    PLATINUM  NEEDLES 

A,  a  straight  needle;  B,  an  ordinary  loop;  C,  a  standard  loop  (the  bend  indi- 
cates the  depth  to  which  the  loop  is  dipped  in  the  liquid  each  time  so  as  to 
remove  equal  amounts) ;  D,  Ravenel's  needle 

Platinum  needles.  The  bacteria  are  transferred  from  one 
culture  vessel  to  another,  or  are  handled  by  means  of  platinum 
needles.  These  are  made  of  No.  25  or  27  gauge  platinum  wire 
for  ordinary  work.  For  special  purposes  lighter  or  heavier 
wire  is  used.  A  piece  50  mm.  long  is  fused  into  a  glass  rod 
or  tube  about  180  mm.  long  and  2.0-2.5  mm.  diameter.  The 
danger  of  the  glass  cracking  at  the  point  of  insertion  of  the 
wire  when  the  needle  is  heated  can  be  lessened  by  fusing  on 
the  end  of  the  ordinary  glass  rod  a  bead  of  readily  fusible 
glass,  into  which  the  wire  is  inserted.  Jena  glass,  No.  397, 
III,  is  excellent  for  this  purpose. 


CULTURAL  TECHNIQUE  31 

All  trouble  can  be  avoided  by  the  use  of  the  needle  handle 
devised  by  Kavetiel.  An  aluminum  rod  of  2.5-3  mm.  diameter 
is  procured  and  cut  into  pieces  150-200  mm.  long.  In  the  end 
of  each  rod  a  hole  is  drilled  with  a  small  drill.  The  platinum 
wire  is  inserted 'into  the  hole  and  the  end  of  the  rod  ham- 
mered lightly  so  as  to  close  the  hole  about  the  wire.  The 
aluminum  rod  may  be  inserted  in  a  piece  of  glass  tubing,  so 
that  it  can  be  more  easily  handled. 

Each  student  should  have  two  inoculating  needles,  (1)  one 
in  which  the  wire  is  straight  and  is  used  for  puncture  inocu- 
lations, (2)  and  another  in  which  a  loop  is  formed  at  the  free 
end,  which  is  used  in  making  transfers  from  liquid  cultures. 
The  loop  should  be  made  around  an  eight-penny  common 
nail  which  has  a  gauge  of  10 J.  The  needle  must  always  be 
heated  to  red  heat  for  its  entire  length,  and  the  lower  part 
of  the  glass  rod  be  passed  through  the  flame  several  times  in 
order  to  sterilize  the  needle  before  use.  The  wire  should  be 
allowed  to  cool  for  a  few  seconds  before  it  is  used.  After 
using,  it  must  be  again  heated  to  red  heat  to  sterilize  it  before 
being  laid  down.  The  habit  of  heating  immediately  before 
and  after  using  must  be  acquired,  so  as  to  become  practically 
an  involuntary  act.  If  this  habit  is  acquired,  much  subse- 
quent trouble  will  be  avoided.  The  lower  part  of  the  glass 
should  not  be  soiled  with  media.  A  "loopful"  means  all  the 
fluid  the  loop  can  hold.  This  should  be  a  biconvex  mass,  not 
merely  a  film  covering  the  lumen  of  the  loop. 

Exercise.    Each  student  will  prepare  a  needle  and  a  standard  loop. 

Plate  cultures.    In  testing  the  purity  of  a  culture  or  in  the. 
isolation  of  the  forms  present  in  a  mixture  of  bacteria  plate 
cultures  must  be  prepared.    This  is  done,  in  case  a  culture  is 
to  be  tested  for  its  purity,  by  melting  three  agar  or  gelatin 


32  EXPERIMENTAL   DAIRY  BACTERIOLOGY 

tubes,  cooling  to  42°-45°  C.,  and  marking  them  1,  2,  and  3. 
With  the  sterile  needle  transfer  a  small  amount  of  the  growth 
to  be  tested  to  the  first  tube  of  melted  medium,  replace  the 
plug,  sterilize  the  needle,  and  mix  the  material  by  rotating  the 
tube  between  the  hands.  From  this  tube  transfer  three  loop- 
fuls  to  the  second  tube,  mix,  and  again  carry  three  -loopfuls  to 
the  third  tube.  Pour  each  tube  into  a  sterile  Petri  dish,  spread, 
and  allow  the  medium  to  solidify.  Incubate.  On  one  of  the 
three  dishes  will  usually  be  found  such  a  number  of  colonies 
that  they  will  be  able  to  attain  the  maximum  size  and  devel- 
opment. When  preparing  plates  the  probable  number  of  liv- 
ing organisms  present  must  be  considered  and  the  amount  of 
original  material  taken,  and  the  number  of  loopfuls  trans- 
ferred from  one  tube  to  another  should  be  taken  accord- 
ingly. Three  plates  should  always  be  made.  An  effort  to 
economize  in  material  by  making  one  or  two  plates  is  very 
apt  to  end  in  failure.  Sometimes  one  tube  of  medium  may 
be  saved  by  using  a  water  blank  for  the  first  transfer,  as  this 
culture  is  generally  so  abundantly  seeded  that  it  is  worthless 
for  study. 

Study  of  plate  cultures.  A  macroscopical  and  microscop- 
ical examination  of  the  colonies  appearing  on  the  plates  should 
be  made,  as  the  general  appearance  is  more  or  less  constant 
and  characteristic  for  each  kind  of  bacteria.  The  microscop- 
ical examination  should  be  made  by  inverting  the  Petri  dish 
on  the  stage  of  the  microscope  and  studying  the  colonies  with 
a  low  power  (16  mm.  objective).  The  following  points  should 
be  noted  in  the  examination :  surface  colonies,  i.e.  colonies 
f  which  are  wholly  or  partially  on.  the  surface  of  the  medium : 
form  of  colony ;  size  of  colony ;  surface  elevation ;  topography 
of  surface ;  microscopic  internal  structure  of  colony ;  micro- 
scopic structure  of  edge  of  colony ;  color  determined  both  by 


CULTURAL  TECHNIQUE  33 

transmitted  and  reflected  light.  Specific  or  technical  terms  are 
used  to  describe  these  characters.  These  the  student  should 
learn  at  an  early  date,  so  as  to  acquire  habits  of  precision  in 
the  matter  of  species  description.  In  the  examination  of  deep 
colonies,  i.e.  imbedded  in  the  medium,  note  the  form,  size, 
microscopic  structure,  consistency,  color,  change  in  surround- 
ing medium.  Deep  colonies  as  a  rule  are  far  less  character- 
istic than  the  superficial  ones. 

Test-tube  cultures.  The  pure  cultures  are  maintained  and 
are  studied  in  "  tube  cultures."  The  transfers  are  made  by 
means  of  the  platinum  needles  from  the  colony  on  the  plate 
culture  to  tubes  containing  various  kinds  of  media.  A  large 
part  of  the  detailed  study  of  an  organism  consists  in  noting 
the  character  of  the  growth  and  the  changes  produced  in 
various  media  in  the  absence  of  all  other  forms,  i.e.  in  pure 
culture,  by  the  organism  in  question. 

Fishing.  In  the  preparation  of  test-tube  cultures  from  the 
plate  cultures  care  should  be  taken  in  the  selection  of  the 
colony  from  which  the  tube  culture  is  to  be  inoculated. 
The  plate  should  be  examined  macroscopically  and  a  ring 
drawn  with  a  colored  wax  pencil  about  each  colony  to  be 
used.  The  colony  and  its  immediate  neighborhood  should 
then  be  examined  under  the  low  power  of  the  microscope, 
in  order  to  determine  whether  there  are  any  near-by  micro- 
scopic colonies.  Mark  the  colonies  which  show  a  clear  zone 
of  several  millimeters  in  width  about  them.  Sterilize  the 
platinum  needle,  remove  the  cover  of  the  Petri  dish,  and 
touch  the  colony  with  the  end  of  the  needle.  With  the  in- 
fected needle  inoculate  the  test-tube  culture ;  mere  contact 
of  the  inoculating  needle  with  the  surface  of  the  colony  is 
amply  sufficient  to  obtain  cells  enough  to  seed  the  new  cul- 
ture. It  is  not  necessary  to  transfer  a  visible  amount  of 


34  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

growth.  From  the  tube  culture  after  the  incubation  inocu- 
lations can  be  made  into  various  media. 

Liquid  cultures.  These  are  made  in  nutrient  broth, 
milk,  etc. 

Stab  cultures  are  made  in  solid  gelatin  and  agar  by  in- 
fecting the  end  of  the  needle  with  a  bit  of  the  growth  from 
the  colony  or  previous  culture,  and  thrusting  the  needle  for 


INOCULATING  TUBE  CULTURES 


The  cotton  plugs  should  be  held  in  such  a  manner  that  the  lower  part  will  not 
come  in  contact  with  the  hand,  and  so  they  can  be  easily  replaced 

nearly  its  entire  length  into  the  medium  and  withdrawing. 
The  gelatin  should  not  crack  on  insertion  of  the  needle ;  if 
it  does  so,  a  fresh  tube  must  be  melted  in  warm  water  and 
resolidified  in  cold  water.  The  cracking  is  due  to  the  drying 
out  of  the  upper  layers  of  the  gelatin  in  the  tube. 

The  culture  of  the  organism  must  be  kept  pure.    During 
the  various  manipulations  the  cultures  are  constantly  exposed 


CULTURAL  TECHNIQUE  35 

to  contamination  from  the  air  and  from  the  surrounding 
objects.  All  operations  must  be  carried  out  in  such  a  way 
as  to  permit  no  contamination  to  take  place.  In  inoculating 
a  culture  tube  the  cotton  plug  must  be  removed  and  held 
in  such  a  way  that  the  portion  which  is  inserted  into  the 
tube  shall  not  come  in  contact  with  any  object.  The  tube 
or  tubes  should  be  held  as  illustrated  in  Fig.  8.  If  any 
cotton  adheres  to  the  mouth  of  the  tube,  it  should  be  re- 
moved by  passing  the  tube  through  the  flame  of  a  Bunsen 
burner.  The  tubes  should  be  kept  open  no  longer  than  neces- 
sary, and  the  needle  sterilized  just  before  using. 

Streak  cultures.  Agar  may  be  allowed  to  solidify  with  the 
tubes  in  an  inclined  position,  thus  giving  a  large  sloping  sur- 
face that  is  inoculated  by  drawing  over  it  in  a  single  stroke 
the  infected  needle  or  loop.  Care  should  be  used  not  to  cut 
or  break  the  surface  of  the  medium.  Potato,  blood  serum,  and 
egg  are  used  in  the  same  manner.  Gelatin  can  be  used  in 
this  way  for  organisms  that  are  unable  to  liquefy  it. 

Study  of  test-tube  cultures.  The  following  points  should 
be  noted  in  the  tube  cultures; 

Nutrient  broth :  conditions  of  fluid,  —  whether  clear  or 
turbid  ;  character  of  turbidity ;  amount  of  sediment ;  surface 
pellicle;  odor. 

Gelatin  stab  cultures  :  character  of  growth  along  line  of  in- 
oculation ;  surface  growth ;  extent  of  liquefaction ;  shape  of 
liquefied  area. 

Agar  stab  cultures :  character  of  growth  along  line  of  in- 
oculation ;  surface  growth. 

Streak  cultures :  form ;  size ;  surface  elevation ;  topography 
of  surface ;  color ;  consistency ;  odor ;  luster ;  change  hi  medium. 

For  list  and  definition  of  the  terms  employed  in  the  de- 
scription of  the  cultural  characters  of  an  organism,  see  Frost's 


36  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Laboratory  Bacteriology,  and  Chester's  Determinative  Bacteri- 
ology, and  also  Appendix  B. 

Exercise.  Each  student  will  prepare  from  the  mixed  culture  fur- 
nished him  a  set  of  plates,  using  agar  as  the  culture  medium,  and  will 
isolate  the  different  organisms  in  pure  culture  and  make  a  detailed 
cultural  study  of  the  same.  From  the  different  types  of  colonies  on 
the  agar  plates  inoculate  agar  slopes.  Incubate  these  from  twenty- 
four  to  forty -eight  hours,  select  two  of  each  type  for  further  study, 
making  agar-stab,  gelatin-stab,  and  nutrient-broth  cultures  from 
each  ;  also  prepare  gelatin  plates. 


CHAPTER  II 

MICROSCOPICAL  TECHNIQUE 

The  student  of  bacteriology  should  be  familiar  with  the 
use  of  the  compound  microscope.  If  experience  with  this 
instrument  has  been  gained  in  other  fields  of  biology,  the 
peculiar  methods  of  bacteriology  are  easily  acquired.  If,  how- 
ever, the  student  has  had  no  previous  training,  he  should 
familiarize  himself  with  the  construction  and  purposes  of  the 
different  parts  of  the  microscope  by  a  perusal  of  the  book- 
lets issued  by  various  optical  companies  on  the  use  and  care 
of  the  microscope,  and  by  a  study  of  the  first  three  chapters 
in  Winslow's  Elements  of  Applied  Microscopy. 

The  microscope  and  its  accessories.  The  microscope  for 
bacteriological  purposes  should  be  provided  with  both  a  coarse 
and  a  fine  adjustment,  with  three  objectives, — 16  mm.(|  inch), 
4  mm.  (\  inch),  and  2  mm.  (^  inch),  —  equivalent  focus,  two 
oculars, —  25  mm.  (1  inch)  and  50  mm.  (2  inch), — a  triple  nose 
piece,  so  that  the  objectives  can  be  easily  changed,  and  an  Abbe 
substage  condenser,  with  iris  diaphragms  both  above  and  be- 
low the  lens  of  the  condenser.  The  Abbe  condenser  and  the 
yV-inch  oil-immersion  lens  are  indispensable  to  the  ordinary 
equipment  of  a  compound  microscope  for  bacteriological  work. 

In  the  examination  of  bacteria  high  powers  of  magnifica- 
tion must  be  used  (500-800  diameters).  In  order  to  secure 
these  magnifications  very  small  lenses  must  be  employed, 
thus  restricting  the  amount  of  light  passing  through  them. 
The  modifications  of  the  ordinary  compound  microscope,  to 

37 


FIG.  9.    SECTION  or  A  MICROSCOPE 

01,  object ;  02,  real  image  in  F2,  transposed  by  the  collective  lens,  to  Os,  real 
image  in  eyepiece  diaphragm;  04,  virtual  image  formed  at  the  projection  dis- 
tance C,  250  mm.  from  EP,  eye  point ;  CD,  condenser  diaphragm ;  L,  mechan- 
ical tube  length  (160  mm.) ;  1,  2,  3,  three  pencils  of  parallel  light  coming  from 
different  points  of  a  distant  illuminant,  for  instance,  a  white  cloud,  which  il- 
luminate three  different  points  of  the  object.  (After  Bausch  &  Lomb  Optical  Co.) 

38 


MICROSCOPICAL  TECHNIQUE  39 

adapt  it  to  bacteriological  work,  are  for  the  purpose  of  concen- 
trating the  light,  and  thus  enable  higher  power  objectives  to 
be  used. 

Abbe  condenser.  The  purpose  of  the  condenser  is  to  collect 
a  large  number  of  rays  of  light  and  bring  them  to  a  focus  in 
the  plane  of  the  object  to  be  examined.  The  plane  mirror 
should  always  be  used  with  natural  light ;  the  concave  mirror 
with  artificial  light.  The  condenser  may  be  focused  by  means 
of  the  screw  beneath  the  stage  of  the  microscope. 

Diaphragm.  The  purpose  of  the  diaphragm  is  to  regulate 
the  amount  of  light  reflected  by  the  mirror  through  the  con- 
denser. For  stained  preparations  of  bacteria  the  iris  dia- 
phragm must  be  open.  In  the  examination  of  unstained 
and  living  forms  the  light  should  be  diminished  by  partial 
closure  of  the  diaphragm,  so  as  to  emphasize  the  slight  vari- 
ations in  density  of  the  protoplasm. 

Light.  The  mirror  should  be  so  adjusted  as  to  illuminate 
the  field  evenly  when  viewed  through  the  tube  of  the  micro- 
scope with  the  ocular  removed.  North  light  is  to  be  preferred, 
and  direct  sunlight  should  never  be  used.  The  light  coming 
from  a  white  cloud  or  the  clear  sky  is  best. 

Focusing.  It  should  be  a  rule  to  work  with  as  low  a  power 
as  possible,  so  as  to  cover  as  large  a  field  as  is  permissible. 
Always  begin  the  examination  of  preparations  of  bacteria 
with  a  4-mm.  objective  and  a  2-inch  ocular,  using  the  2-mm. 
oil-immersion  objective  for  a  more  detailed  examination. 

The  proper  objective  should  be  turned  into  place,  and  with 
the  eye  held  at  the  side  of  the  stage,  the  tube  should  be 
lowered  carefully  by  the  use  of  the  coarse  adjustment  until 
the  lens  is  nearly  in  contact  with  the  cover  glass  of  the  prepa- 
ration. With  the  eye  at  the  ocular,  then  focus  slowly  upward 
with  the  fine  adjustment,  meanwhile  moving  the  preparation 


40 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


slightly  with  the  fingers.    With  high-power  lenses  never  focus 
down  with  the  eye  at  the  ocular. 

Oil-immersion  objective.  The  rays  of  light  passing  obliquely 
through  glass  into  air  are  refracted  away  from  the  perpen- 
dicular ;  or,  in  other  words,  the  rays  of  light  entering  the 
lens  of  the  microscope  are  less  in  number  than  if  there  were 
no  difference  between  the  indices  of  refraction  of  glass  and 


EIG.  10.    PRINCIPLE  OF  THE  OIL-!MMERSION  LENS 
L,  lens ;  2),  object  slide ;  Od  and  Ob,  rays  of  light.    (After  Hager-Mez) 

air.  By  placing  a  liquid  having  the  same  index  of  refraction 
as  glass  between  the  lens  and  the  object,  the  amount  of 
light  entering  the  lens  is  increased,  and  as  far  as  the  amount 
of  light  is  concerned,  the  object,  the  slide,  the  lens,  and  the 
intervening  liquid  act  as  a  single  piece  of  glass.  The  immer- 
sion liquid  used  is  cedar  oil,  of  such  a  degree  of  concentration 
as  to  have  the  same  index  of  refraction  as  glass.  If  the  oil 
becomes  thickened  by  evaporation  of  the  solvent,  the  bottle 
should  be  cleaned  and  a  fresh  supply  obtained. 


MICROSCOPICAL  TECHNIQUE  41 

In  using  the  immersion  lens  a  drop  of  cedar  oil  is  placed 
on  the  cover  glass,  care  being  taken  to  avoid  air  bubbles. 
The  tube  of  the  microscope  is  lowered  until  the  lens  touches 
the  drop  and  is  nearly  in  contact  with  the  cover  glass.  This 
must  be  done  with  care,  for  if  the  tube  is  lowered  by  the 
coarse  adjustment  until  the  objective  touches  the  cover  glass, 
a  broken  preparation  or  a  damaged  lens  is  likely  to  result. 
In  some  of  the  modern  types  of  microscopes  the  fine  adjust- 
ment is  so  arranged  that  its  action  is  stopped  when  the 
objective  touches  the  preparation. 

At  the  end  of  the  day's  work  the  oil  must  be  removed 
from  the  lens  by  wiping  with  Japanese  lens  paper,  which 
should  be  kept  in  an  envelope  or  glass  jar  to  protect  it  from 
dust.  If  the  lens  becomes  sticky  by  the  drying  of  the  oil, 
the  latter  must  be  removed  by  saturating  a  piece  of  lens 
paper  with  xylene  and  wiping  the  lens  carefully.  An  excess 
of  xylene  must  be  avoided,  as  it  is  a  solvent  of  the  lens 
mounting.  After  the  treatment  the  lens  should  be  dried 
with  clean  paper. 

Slides  and  cover  glasses.  The  American  slide  is  25  x 
75  mm.  of  clear  white  glass.  The  cover  glasses  used  for  bac- 
teriological work  must  be  thin  on  account  of  the  short  work- 
ing distance  of  the  higher-power  objectives.  No.  2  cover 
glasses  are  usually  employed,  having  a  thickness  of  0.17  — 
0.25  mm.  The  slides  may  be  cleaned  by  washing  in  water  or 
alcohol  and  drying  with  a  towel.  The  cover  glasses  must  be 
wholly  free  from  fat,  in  order  that  an  even  distribution  of  the 
liquid  to  be  examined  may  be  obtained.  New  cover  glasses  are 
cleaned  by  washing  in  water,  and  then  immersing  in  alcohol 
to  which  3  per  cent  of  hydrochloric  acid  has  been  added.  The 
cover  glasses  are  dried  by  rubbing  between  driers  made  of 
wooden  blocks  200  X  100  X  25  mm.,  covered  with  several 


42  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

layers  of  cotton  cloth.  The  glass  covers  are  then  placed  on  a 
flat  piece  of  Russia  iron  and  heated  over  a  Bunsen  flame  for 
one  hour.  The  iron  should  not  become  red-hot.  The  prolonged 
heating  destroys  all  traces  of  fat.  The  clean  covers  should  be 
handled  with  forceps,  for  if  touched  with  the  fingers  a  film 
of  grease  is  left.  If  a  drop  of  water  is  placed  on  a  perfectly 
clean  cover  glass,  it  will  spread  in  a  film  over  the  entire  sur- 
face. Store  the  cover  glasses  in  a  clean  Petri  dish. 

Slides  and  covers  that  have  been  used  may  be  cleaned  by 
placing  them  in  turpentine  to  remove  the  oil,  then  in  a  clean- 
ing fluid  composed  of  20  grams  of  potassium  bichromate, 
100  cc.  of  water,  100  cc.  of  sulphuric  acid.  They  should  be 
digested  in  this  mixture  until  the  organic  matter  is  destroyed. 
Rinse  until  free  from  the  cleaning  mixture,  and  then  treat  as 
new.  The  slides  and  covers  may  also  be  cleaned  by  placing 
in  alcohol  and  adding  2—3  volumes  of  concentrated  nitric 
acid,  allowing  them  to  stand  in  this  for  some  time,  rinsing, 
rubbing  to  remove  any  organic  matter  not  destroyed,  and 
wiping  from  alcohol.  The  addition  of  the  acid  should  be 
made  out  of  doors  or  in  a  hood,  on  account  of  the  fumes 
given  off. 

Exercise.    Clean  |  ounce  of  cover  glasses  and  50  slides. 

Staining  solutions.  The  size  and  transparency  of  the  bac- 
teria make  their  differential  study  difficult  in  unstained  prep- 
arations. In  order  that  their  morphology  may  be  more  readily 
determined,  the  use  of  stains  or  dyes  is  resorted  to.  The  stains 
usually  employed  are  the  basic  aniline  dyes.  For  ordinary 
purposes  methylene  blue,  fuchsin,  gentian  violet,  and  bismarck 
brown  are  sufficient. 

The  stock  solutions  kept  in  the  laboratory  are  made  by 
preparing  a  saturated  solution  of  the  various  dyes  in  95  per 


MICROSCOPICAL  TECHNIQUE  43 

cent  alcohol.  These  stock  solutions  keep  well,  but  cannot  be 
used  for  staining  purposes.  From  them  the  solution  used  is 
prepared  by  dilution  with  distilled  water,  and  filtering  the 
mixture. 

1.  Aqueous  solution  of  fuchsin  is  made  by  adding  to  95  cc. 
of  distilled  water  5  cc.  of  the  saturated  alcoholic  solution. 

2.  Aqueous    solution    of    methylene    blue    is    similarly 
prepared. 

3.  Aqueous  solution  of  gentian  violet  is  similarly  prepared. 

4.  Loeffler's,  or  alkaline  methylene  blue,  is  made  by  mixing 
30  cc.  of  saturated  alcoholic  solution,  1  cc.  of  a  1  per  cent 
potassium  hydrate  solution,  and  100  cc.  of  distilled  water. 

5.  Carbol-fuchsin  is  made  by  adding  to  a  5  per  cent  solu- 
tion of  carbolic  acid  a  saturated  alcoholic  solution  of  fuchsin 
until  a  metallic  luster  is  noted  on  the  surface  of  the  liquid. 

6.  Aniline-water  gentian  violet  is  prepared  as  follows :  To 
98  cc.  of  distilled  water  are  added  2  cc.  of  aniline  oil.    Shake 
vigorously.    Filter,  and  to  75  cc.  add  25  cc.  of  saturated  alco- 
holic solution  of  gentian  violet.    The  solution  should  be  re- 
filtered  after  standing  twenty-four  hours,  to  remove  drops  of 
oil  that  may  have  settled  out. 

7.  Iodine  solution  (Lugol's  or  Gram's  solution)  is  prepared 
as  follows :  One  gram  of  iodine  is  dissolved  in  a  solution  made 
of  2  grams  of  potassium  iodide  in  300  cc.  of  distilled  water. 

These  staining  solutions  keep  well  with' the  exception  of 
aniline-water  gentian  violet,  which  must  be  renewed  at  fre- 
quent intervals. 

Exercise.  From  the  saturated  alcoholic  solutions  furnished,  pre- 
pare 50  cc.  of  an  aqueous  solution  of  methylene  blue,  gentian  violet, 
aniline-water  gentian  violet,  carbol-f  uchsin,  and  iodine  solution.  Place 
them  in  dropping  bottles,  or  in  bottles  provided  with  corks  through 
which  a  piece  of  small  glass  tubing  is  passed,  by  which  small  quan- 
tities of  the  solution  can  be  removed  without  spilling. 


44  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Making  of  stained  preparations.  Most  forms  of  bacteria 
found  in  milk  and  its  products  can  be  stained  by  the  use 
of  the  simple  aqueous  stains.  The  staining  is  carried  out  as 
follows:  A  clean  cover  glass  is  removed  from  the  dish  in 
which  they  are  stored,  and  placed  in  a  pair  of  cover-glass 
forceps.  On  the  cover  is  placed  a  very  small  drop  (size  of  a 
pin  head)  of  water  by  means  of  the  platinum  loop,  or  from 
the  glass  tip  of  the  flushing  arrangement.  With  a  sterile 
needle  a  minute  portion  of  the  growth  on  a  solid  medium  is 
removed,  mixed  with  the  drop  of  water,  and  spread  over  as 
large  a  portion  of  the  cover  glass  as  possible.  When  liquid 
cultures  are  to  be  examined,  a  loopful  should  be  removed  and 
spread  over  the  cover  glass.  The  preparation  should  be  al- 
lowed to  air  dry.  If  subjected  to  the  action  of  heat,  the  indi- 
vidual organisms  are  heaped  up  in  concentric  masses,  instead 
of  being  uniformly  distributed.  The  preparation  may  be  made 
on  the  slide  itself,  thus  eliminating  the  use  of  the  cover  glass. 

In  order  to  cause  the  bacteria  to  adhere  to  the  glass  and 
not  be  washed  off  in  subsequent  operations,  the  preparation 
must  be  fixed  by  passing  it  rapidly  through  the  upper  part 
of  a  Bunsen  flame  three  times  (film  side  up).  With  nearly 
all  forms  this  is  sufficient  to  cause  the  coagulation  of  the  pro- 
teids  of  the  cell  and  thus  permit  of  adhesion  to  the  glass. 
When  difficulty  is  experienced  in  fixing  the  preparations  in 
this  way,  a  couple  of  drops  of  95  per  cent  alcohol  may  be 
allowed  to  evaporate  from  the  preparation  during  the  ordi- 
nary fixing  process. 

The  forceps  with  the  cover  glass  or  the  slide  are  placed 
on  the  staining  dish  and  the  preparation  flooded  with  the 
stain,  which  is  allowed  to  act  from  one  to  five  minutes,  after 
which  it  is  washed  off  with  a  stream  of  distilled  water.  The 
wet  cover  glass  is  taken  in  a  pair  of  fine-pointed  forceps  and 


MICROSCOPICAL  TECHNIQUE  45 

placed  film  side  down  on  a  clean  slide,  avoiding  air  bubbles 
by  allowing  first  one  side  of  the  cover  glass  to  come  in  con- 
tact with  the  slide,  and  then  gradually  lowering  the  cover 
glass  into  place.  The  excess  of  water  is  removed  and  the 
upj>er  surface  of  the  cover  glass  dried  by  blotting  with  filter 
paper.  The  preparation  is  now  ready  to  be  examined  directly, 
or  a  permanent  preparation  may  be  made  by  allowing  the 


FIG.  11.    STAINING  DISH  AND  DROPPING  BOTTLE  FOR  STAINS 

An  enameled-ware  basin  covered  with  J-inch-mesh  galvanized 
wire  cloth 

cover  glass  to  air  dry  after  staining  and  washing.  On  a  slide 
place  a  small  drop  of  Canada  balsam  dissolved  in  xylene,  and 
then  lower  the  cover-glass  preparation,  film  side  down,  on  to 
the  drop.  The  balsam  should  be  of  such  consistency  that  it 
will  spread  under  the  weight  of  the  cover  glass,  and  should 
be  present  in  such  quantities  that  it  will  fill  the  space  be- 
tween the  cover  glass  and  slide  to  the  edge  of  the  cover, 
but  not  run  beyond.  The  preferable  method  is  to  make  a 
preliminary  examination  of  the  preparation  in  the  water 


46  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

mount.  If  it  is  then  desired  to  make  a  permanent  mount, 
allow  the  preparation  to  dry,  when  the  cover  will  fall  from  the 
slide  when  the  latter  is  gently  tapped.  The  cover  can  then  be 
mounted  in  "balsam.  Allow  the  balsam  to  become  dry  before 
examining  again  (which  process  takes  several  days),  as  other- 
wise the  cover  glass  is  likely  to  be  displaced.  Preparations 
made  on  the  slide  may  be  drained,  carefully  blotted  with 
filter  paper,  and  allowed  to  become  perfectly  dry  before  ex- 
amining, as  the  immersion  oil  is  placed  directly  on  the  prep- 
aration. If  it  is  desired  to  make  a  permanent  mount  of  such 
a  preparation,  the  immersion  oil  is  removed  by  the  use  of 
xylene,  a  drop  of  balsam  placed  on  the  slide,  and  a  cover 
glass  placed  in  position. 

When  a  preparation  is  properly  made  from  a  surface 
growth  on  solid  media,  the  bacteria  should  appear  as  though 
suspended  in  air,  i.e.  the  field  of  the  microscope  should  be 
perfectly  clear  and  the  bacteria  evenly  distributed  in  the 
field.  The  common  error  is  the  use  of  too  much  growth,  re- 
sulting in  a  thick  film  in  which  the  bacteria  are  crowded 
together  and  piled  up  in  such  a  manner  as  to  make  a  satis- 
factory study  impossible. 

Exercise.  Make  cover-glass  preparations  from  the  cultures  fur- 
nished you,  using  methylene  blue  or  alkaline  methylene  blue  for 
preparations  from  milk,  broth,  and  gelatin  cultures,  gentian  violet 
for  preparations  from  agar. 

Examination  of  stained  preparations.  In  the  examination 
of  cover-glass  preparations  the  following  points  should  be 
noted:  (1)  the  form  of  the  bacteria,  whether  spherical,  elon- 
gated, or  spiral;  (2)  the  grouping  of  the  cells,  whether  iso- 
lated, in  long  or  short  filaments,  spherical  cells  in  plane 
surfaces,  in  irregular  masses,  or  in  packets ;  (3)  the  in- 
ternal structure  of  the  cell,  in  both  faintly  and  deeply  stained 


OF 

a 

ICROSCOPICAL  TECHNIQUE  47 

preparations,  the  presence  of  vacuoles,  capsules,  spores,  the 
ease  with  which  the  bacteria  take  the  simple  aqueous  stains. 
Gram's  stain.  Some  stains  are  of  value  in  that  they  not 
only  bring  out  the  morphology  of  the  organism,  but  also  have 
a  diagnostic  value  in  differentiating  one  species  from  another. 
The  one  most  frequently  used  for  this  purpose  is  the  Gram 
stain.  The  staining  of  the  preparation  is  as  follows :  Prepare 
and  fix  the  preparation  as  previously  described,  and  then  stain 
for  one  minute  with  aniline-water  gentian  violet.  Wash  the 
preparation  and  then  flood  with  iodine  solution,  leaving  it 
in  contact  with  the  preparation  for  two  minutes.  Flood  with 
95  per  cent  alcohol,  repeating  this  operation  until  no  more 
color  is  given  off.  Wash,  mount  in  water,  and  examine. 
Organisms  which  retain  the  stain  are-  said  to  be  Gram  posi- 
tive, all  others  Gram  negative. 

Exercise.  Stain  preparations  from  the  cultures  furnished  by 
Gram's  method. 

Flagella  stain.  The  staining  of  the  delicate  flagella  is  a 
difficult  process.  No  one  method  is  always  successful.  The 
number  and  arrangement  of  the  flagella  are  of  diagnostic 
value.  The  following  method  (Bunge's)  is  one  of  the  best  in 
use :  A  mordant,  which  so  alters  the  protoplasm  of  the  cell 
as  to  make  it  more  readily  stained,  is  prepared  by  mixing 
25  cc.  of  a  5  per  cent  aqueous  solution  of  ferric  chloride  with 
75  cc.  of  a  saturated  aqueous  solution  of  tannic  acid. 

From  an  eighteen-hour  to  a  twenty-four-hour  culture  of 
the  organism  on  agar  remove  a  small  amount  of  the  growth 
and  place  in  a  large  drop  of  tap  water.  Do  not  mix,  but 
allow  the  organisms  to  diffuse  themselves,  as  mixing  with 
the  needle  breaks  off  many  of  the  flagella.  After  standing 
a  few  moments  remove  a  loopful  to  a  perfectly  clean  cover 
glass  and  allow  the  drop  to  spread  of  itself.  Dry,  and  fix 


48  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

with  extreme  care,  to  avoid  overheating,  by  holding  the 
cover  glass  in  the  fingers  and  passing  through  the  flame. 
Flood  the  cover  glass  with  the  mordant  and  heat  over  the 
steam  from  a  water  bath  for  two  minutes.  Wash  off  the  mor- 
dant and  stain  for  two  minutes  with  carbol-fuchsin,  placing 
the  cover  over  the  steam.  Wash,  dry,  and  mount  in  balsam. 
The  number  and  location  of  the  flagella  should  be  noted. 

Exercise.  From  the  cultures  furnished  make  stained  preparations. 
Determine  the  presence  or  absence  of  flagella. 

Capsule  stain..  In  certain  culture  fluids  containing  al- 
bumen or  sugar  many  organisms  produce  a  more  or  less 
thickened  cell  wall.  The  inner  side  of  the  cell  wall  appears 
as  a  sharp  line,  while  the  outer  is  less  sharp  and  at  times 
is  not  to  be  distinguished.  The  cell  wall  absorbs  a  large 
amount  of  water  and  forms  a  jellylike  substance.  In  the 
stained  preparation  the  cell  contents  appear  deeply  stained, 
the  wall  and  swollen  outer  layer  very  faintly  stained,  or  un- 
stained. The  organisms  appear  as  though  surrounded  by  a 
clear  zone, —  the  capsule.  Such  organisms  are  of  importance 
in  dairy  bacteriology,  since  they  are  frequently  one  of  the 
causes  of  an  abnormal  fermentation  of  milk  and  cream,  the 
so-called  "  slimy  "  or  "  ropy  "  fermentation. 

A  special  stain  may  be  used  to  determine  the  presence  of 
the  capsule.  Prepare  the  film  without  the  use  of  water,  air 
dry,  fix,  apply  glacial  acetic  acid,  and  drain  off  at  once  ;  with- 
out washing,  apply  carbol-fuchsin,  renewing  several  times  to 
remove  the  acid ;  wash  in  a  1-2  per  cent  salt  solution ;  ex- 
amine in  salt  solution.  The  glacial  acetic  acid  fixes  the  muci- 
laginous capsule  so  that  it  does  not  dissolve  in  water. 

Exercise.  Examine  by  the  above  method  the  cultures  furnished  you. 

Spore  stain.  The  presence  or  absence  of  spores  cannot 
be  determined  by  staining  in  all  cases.  Vacuoles,  fat  drops, 


MICROSCOPICAL  TECHNIQUE  49 

crystals,  may  be  mistaken  for  spores  in  preparations  stained 
with  simple  aqueous  dyes.  A  special  stain  may  be  employed 
as  follows :  Place  the  dried  and  fixed  films  in  chloroform  for 
two  minutes,  and  then  in  a  5  per  cent  solution  of  chromic  acid 
for  two  minutes ;  wash  in  water,  cover  with  carbol-fuchsin, 
and  heat  in  the  steam  of  a  water  bath  for  five  minutes.  Wash 
off*  the  stain ;  decolorize  carefully  in  1  per  cent  sulphuric  acid  ; 
wash  in  water;  counterstain  with  methylene  blue  at  room 
temperature  for  ten  seconds;  wash  and  examine. 

The  principle  of  the  process  rests  on  the  peculiar  property 
of  the  spores  towards  stains.  They  stain  with  difficulty,  but, 
when  once  stained,  retain  the  color.  Thus  the  first  stain  can 
be  removed  from  the  cell  with  a  weak  acid,  and  the  cell  then 
counterstained  with  a  differential  dye.  The  position  of  the 
spores  in  the  cells  should  be  noted,  whether  medium  or  ter- 
minal ;  their  shape,  whether  oval  or  round ;  their  size  in  rela- 
tion to  the  mother  cell,  whether  greater  in  diameter  than  the 
mother  cell,  or  of  less  diameter ;  the  presence  of  free  spores, 
i.e.  outside  of  the  mother  cell. 

The  only  method  by  which  the  spores  may  be  identified 
as  such  in  case  of  doubt  is  by  observing  their  germination. 
This  also  is  often  of  diagnostic  value,  and  can  be  observed  in 
the  hanging  block  suggested  by  Hill. 

Exercise.    Make  spore  stains  from  cultures  furnished  you. 

Examination  of  living  bacteria.  The  examination  of  the 
bacteria  in  a  living  condition  will  give  much  information  of 
morphological  value.  The  method  of  procedure  is  as  follows : 
Place  a  drop  of  liquid  medium  —  broth — on  a  cover  glass  or 
slide,  and  inoculate  it  with  a  particle  of  growth  from  a  solid 
culture,  mix  carefully,  and  transfer  a  very  small  drop  to  a 
cover  glass;  do  not  spread.  Invert  over  the  drop  a  hollow 
ground  slide  around  the  cavity  of  which  has  been  placed  a 


50  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

ring  of  vaseline;  press  down  on  the  cover.  The, vaseline  should 
form  a  perfect  seal,  thus  preventing  evaporation  and  enabling 
a  small  drop  to  be  kept  under  observation  for  hours.  On  ac- 
count of  the  transparency  of  the  bacteria  focusing  is  often 
difficult.  The  drop  should  be  placed  in  the  optical  axis  of  the 
microscope,  which  is  easily  done  by  lowering  the  tube  with 
the  4-mm.  objective  in  place  within  one  eighth  of  an  inch 
of  the  cover  glass,  and  then  bringing  the  drop  under  the  lens. 
With  the  eye  at  the  side,  lower  the  tube  until  the  objective  is 
nearly  in  contact  with  the  cover.  Close  the  diaphragm  until 
the  field  is  light  gray  in  color,  and  focus  very  slowly  upward. 
After  finding  the  organisms  the  slide  should  be  moved  until 


FIG.  12.   A  HANGING-DROP  PREPARATION 

the  edge  of  the  drop  is  found,  as  here  the  organisms  can  be 
observed  to  better  advantage  than  in  the  deeper  portion  of 
the  drop.  The  oil-immersion  lens  may  then  be  used  if  de- 
sirable. The  use  of  artificial  light  renders  the  organisms 
more  easily  visible. 

The  form,  size,  and  grouping  of  the  cells  should  be  noted, 
the  power  of  independent  motion,  the  nature  of  the  motion, 
—  direct,  sinuous,  rotating,  etc.  The  molecular  or  Brownian 
motion  (pedesis)  must  not  be  confounded  with  vital  motion. 
This  molecular  motion  is  a  property  common  to  all  finely 
divided  substances  suspended  in  liquids,  and  consists  of  a 
vibratory  or  trembling  rather  than  a  progressive  motion. 
In  order  that  an  organism  be  classed  as  motile,  the  relative 
positions  of  the  cell  with  reference  to  other  cells  must  change. 

Exercise.  Examine  in  hanging  drop  the  cultures  of  yeast  and  of 
bacteria  furnished  you,  and  also  a  preparation  made  by  suspending 
a  little  India  ink  in  water. 


MICROSCOPICAL  TECHNIQUE  51 

Hanging  block.  It  is  frequently  desirable  to  determine 
the  mode  of  germination  of  bacterial  spores,  and  the  mode 
and  rate  of  fission  of  the  vegetating  cell.  This  is  most  con- 
veniently done  by  the  use  of  Hill's  hanging  block.  Pour 
melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of  one 
eighth  of  an  inch.  Cool  the  agar.  Cut  from  it  with  a  sterile 
knife  a  block  one  quarter  to  one  third  of  an  inch  square,  and 
of  the  thickness  of  the  agar  in  the  dish.  Place  the  block, 
under  surface  down,  on  a  glass  slide,  and  protect  from  dust. 
Prepare  in  sterile  water  a  suspension  of  the  organism  to  be 
examined,  if  it  has  been  grown  on  a  solid  medium,  or,  if  a 
broth  culture  is  used,  spread  the  suspension  on  the  upper  sur- 
face of  the  block  as  though  making  a  cover-glass  smear.  Place 
the  slide  with  the  block  in  the  37°  C.  incubator  for  five  or  ten 
minutes  to  dry.  Then  lay  a  clean  sterile  cover  glass  on  the 
inoculated  surface  of  the  block  in  close  contact  with  it,  avoid- 
ing air  bubbles  as  far  as  possible.  Remove  the  block  from 
the  slide  and  invert  the  same.  With  a  platinum  loop  run  a 
few  drops  of  melted  agar  around  the  edge  of  the  block,  to  fill 
the  angles  between  the  sides  of  the  block  and  the  glass.  This 
seal  prevents  slipping  of  the  block.  The  block  should  again 
be  placed  in  the  incubator  for  five  to  ten  minutes  to  dry.  In- 
vert this  preparation  over  a  moist  chamber  made  by  cement- 
ing a  ring  of  glass  to  an  ordinary  slide.  The  hollow  ground 
slide  cannot  be  used  on  account  of  the  depth  of  the  block. 
The  cover  should  be  sealed  in  place  with  white  wax  or 
paraffin.  Vaseline  softens  at  37°  C.  and  allows  the  cover 
glass  to  shift.  The  preparation  may  now  be  observed  with 
the  -J^-inch  immersion  objective.  • 

Measuring  bacteria.  Measurements  under  the  compound 
microscope  are  made  by  means  of  an  ocular  micrometer,  —  a 
disk  of  glass  which  is  placed  on  the  diaphragm  within  the 


52 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


ocular.  The  glass  bears  a  scale  made  up  of  lines  at  an  equal 
but  not  necessarily  known  distance  apart.  The  scale  of  the 
micrometer  should  lie  in  the  plane  of  the  real  image  formed 
by  the  objective.  The  image  of  the  object  thus  lies  on  the 
scale,  and  may  be  measured  by  it  if  the  value  of  each  di- 
vision of  the  scale  be  determined  in  actual  units  of  measure- 
ment. For  this  purpose  a  stage  micrometer  is  necessary,  —  a 
slide  bearing  a  cover  glass  on  which  is  ruled  a  scale  in  0.1 
and  0.01  mm.  The  ratio  of  ocular  divisions  to  the  image  of 
the  stage  divisions  is  determined  by  focusing  with  the  ocular 
micrometer  in  place  on  the  scale  of  the  stage  micrometer. 
The  lines  of  the  two  micrometers  are  made  parallel  by  rotat- 
ing the  ocular.  Make  the  image  of  any  two  lines  on  the  stage 
micrometer  coincide  with  two  lines  on  the  ocular  microm- 
eter, by  drawing  out  the  tube  of  the  microscope  if  neces- 
sary. From  the  data  thus  obtained  it  is  easy  to  calculate 
the  value  of  each  space  on  the  ocular  micrometer  in  0.01  of 
a  millimeter.  The  ocular  used,  the  objective,  the  number  of 
the  ocular  micrometer,  and  the  tube  length  of  the  microscope 
must  be  recorded  and  also  the  value  of  a  single  space  on  the 
ocular  micrometer  for  the  given  system  of  lenses  in  microns 
(0.001  mm.)  expressed  by  the  Greek  letter  /*. 

Exercise.    Determine  the  value  of  the  ocular  micrometer  and  fill 
out  the  blanks  in  the  following  table  : 


No.  of  microscope- 


Make. 


OBJECTIVE 

TUBE  LENGTH 

VALUE  OF  A  DIVISION 
OF  EYEPIECE  MICROMETER 

16mm. 

4  mm. 

2  mm. 

CHAPTER  III 
CONTAMINATION  OF  MILK 

Milk,  meats,  eggs,  fruits,  etc.,  are  classed  as  perishable  food 
products,  for  unless  extraordinary  means  are  taken  to  preserve 
them,  the  period  during  which  they  can  be  used  as  food  is 
limited.  Of  these  milk  is  the  most  perishable.  The  period 
during  which  it  can  be  kept  is  measured  by  hours  rather 
than  by  days.  The  spoiling  of  these  products  is  due  to  the 
growth  of  bacteria  and  other  organisms  in  and  on  the  surface. 
The  more  perfectly  the  product  is  adapted  as  food  for  organ- 
isms, and  the  greater  the  number  which  gain  entrance  to  it, 
the  shorter  will  be  its  period  of  usefulness.  No  other  food 
product  is  to  be  compared  with  milk  as  a  culture  medium 
for  bacteria,  and,  curiously  enough,  no  other  food  product  is  so 
exposed  to  contamination  during  the  process  of  preparation. 

No  'subject  in  dairy  bacteriology  is  more  fundamentally 
important  than  that  relating  to  milk  contamination,  both  in 
the  factory  and  on  the  farm,  and  most  of  the  problems  relating 
to  the  proper  handling  of  milk  come  back  to  this  question. 

The  contamination  of  milk  may  be  considered  from  three 
standpoints, — hygienic,  economic,  and  what  may  be  called  the 
standpoint  of  general  cleanliness.  From  the  hygienic  stand- 
point are  to  be  considered  the  contamination  with  pathogenic 
bacteria  and  the  production  of  poisonous  and  injurious  sub- 
stances in  the  milk  by  the  growth  of  saprophytic  bacteria. 
The  economic  side  of  the  question  has  to  do  with  the  dimin- 
ished keeping  quality  of  milk  and  of  other  dairy  products, 

63 


54  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

and  is  especially  important  to  the  milk  producer  and  handler, 
and  also  to  the  consumer.  If  in  any  practical  way  the  contam- 
ination may  be  lessened  and  the  keeping  quality  improved, 
the  value  of  the  product  will  be  enhanced.  Entirely  apart 
from  the  economic  and  hygienic  relations  of  the  contamina- 
tion of  milk  may  be  considered  its  relation  to  general  clean- 
liness. Wholesome  food  prepared  in  a  cleanly  manner  is 
desirable,  not  because  unclean  food  may  cause  sickness  or 
may  not  keep  so  long,  but  simply  for  the  sake  of  cleanli- 
ness alone. 

When  it  is  considered  that  milk  is  largely  produced  for 
human  food,  either  in  the  form  of  milk,  or  as  butter  or  cheese, 
it  must  be  admitted  that  the  circumstances  surrounding  its 
production  are,  even  under  the  best  of  stable  conditions,  far 
from  what  would  be  looked  upon  as  desirable  in  the  prepara- 
tion of  any  other  food  product.  Especially  is  this  true  when 
it  is  kept  in  mind  that  meat,  fruits,  vegetables,  etc.,  -^-  food 
products  likely  to  be  soiled  with  dirt,  —  can  be  washed  and 
rendered  clean  and  wholesome.  The  mud,  manure,  and  dust 
which  fall  into  milk  are  partially  dissolved  and  cannot  be 
removed.  Straining  removes  the  grosser  pollution,  such  as 
straw  and  hair,  but  does  not  remove  the  bacteria  which  are 
attached  to  these  objects.  Milk  is  a  food  product  that,  once 
rendered  dirty,  cannot  be  made  clean.  Many  of  the  sources 
of  contamination  are  wholly  preventable,  some  partially,  and 
still  others  to  a  very  slight  extent.  The  student  should  become 
thoroughly  familiar  with  all  the  sources  of  contamination  and 
with  the  different  practical  means  for  overcoming  or  removing 
the  same. 

Contamination  from  barn  air.  Dust  particles  floating  in 
the  air  invariably  carry  a  larger  or  smaller  number  of  bac- 
teria, depending  upon  the  nature  of  the  surface  from  which 


AMINATION  OF  MILK  55 


the  dust  came.  In  the  cow  stable  many  operations  are  carried 
on  that  tend  to  raise  a  large  amount  of  dust,  which  in  many 
cases  comes  from  materials  that  are  very  rich  in  bacterial  life. 
The  dried  manure  which  is  scattered  over  the  floor,  and  in  time 
pulverized,  is  an  important  source.  The  dust  raised  whenever 
cattle  are  brushed  is  largely  made  up  of  manure  particles, 
epidermal  scales,  and  scurf  from  the  skin.  The  operation  of 
feeding  dry  feed  (hay,  corn,  fodder,  etc.)  is  a  source  of  dust, 
for  the  way  in  which*  these  materials  are  harvested  tends  to 
contaminate  them  with  soil  from  the  fields,  and  the  surface 
layers  of  the  soil  are  especially  rich  in  bacterial  life.  The 
dust  raised  during  the  various  barn  operations  is  compara- 
tively heavy,  and  for  the  most  part  soon  settles,  carrying  its 
load  of  germ  life. 

The  relative  extent  and  importance  of  this  source  of  con- 
tamination under  varying  conditions  may  be  determined  as 
follows:  Tubes  of  lactose  gelatin  are  melted,  poured  into 
sterile  Petri  dishes,  and  allowed  to  become  completely  solidi- 
fied. A  number  of  these  plates  are  exposed  in  the  stable 
when  dust  is  present  in  minimum  quantities,  i.e.  at  a  time 
when  no  operation  that  produces  dust  has  been  carried  on 
for  two  or  three  hours.  The  remaining  plates  should  be  ex- 
posed shortly  after  some  dust-producing  operation  has  been 
completed.  This  exposure  should  be  made  by  placing  the 
plates  on  a  level  surface,  removing  the  cover,/  which  is  placed 
right  side  up  by  the  side  of  the  plate,  so  as  not  to  add  a  further 
contamination  when  it  is  replaced.  The  exposures  should  be 
made  in  different  parts  of  the  stable  and  the  plates  so  located 
as  to  be  fully  exposed  to  the  falling  dust,  and  yet  be  protected 
from  dirt  being  thrown  on  to  them.  A  definite  period  of  ex- 
posure should  be  made,  although  this  time  may  vary  within 
reasonable  limits  (thirty  to  one  hundred  and  twenty  seconds). 


56  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

The  plates  should  be  examined  daily  and  the  final  count 
made  after  from  five  to  six  days'  incubation.  Often  the  pres- 
ence of  a  large  number  of  molds  and  liquefying  bacteria  neces- 
sitates a  much  shorter  period  of  incubation.  The  results  may 
be  recorded  in  terms  of  the  number  of  colonies  per  plate,  but 
for  more  graphic  presentation  should  be  converted  into  the 
number  of  organisms  that  would  fall  into  a  12-inch  milk  pail 
during  an  average  milking  period  (five  minutes). 

Exercise.  Each  student  will  prepare  ten  pla'tes  as  described  :  (1)  ex- 
pose four  of  them  in  the  stable  at  a  time  when  dust  is  present  in  mini- 
mum quantities  ;  (2)  four  when  different  barn  operations  producing 
dust  are  in  progress  ;  (3)  the  remaining  plates  are  to  be  exposed  in 
the  immediate  neighborhood  of  an  animal  that  is  being  brushed. 

Contamination  from  coat  of  animal.  The  udder  and  flanks 
of  animals,  even  under  the  best  of  stable  conditions,  are  likely 
to  become  soiled  with  manure,  and  unless  elaborate  precau- 
tions are  taken  to  remove  the  dirt,  it  is  dertain  to  be  an 
important  source  of  bacteria  in  milk,  as  during  the  milk- 
ing process  the  movements  of  the  animal  and  milker  will 
dislodge  dust  and  accompanying  bacteria  not  only  from  the 
udder  proper  but  from  the  flanks  and  sides  of  the  cow.  More 
or  less  of  this  dirt  and  dust  finds  its  way  into  the  open 
milk  pail. 

The  bacterial  content  of  fresh  manure  is  exceedingly  high ; 
hence  very  small  quantities  may  increase  the  germ  content 
of  the  milk  to  a  marked  degree. 

The  importance  of  these  sources  of  contamination  under 
various  conditions  may  be  determined  as  follows :  Plates  are 
prepared  as  in  the  previous  exercise,  and  exposed  under  the 
udder  of  a  cow  while  this  organ  is  being  manipulated  as 
during  the  milking  process.  The  culture  plate  should  be  ex- 
posed by  placing  it  in  a  milk  pail  which  is  held  in  the  same 


CONTAMINATION  OF  MILK  57 

position  as  during  milking.  Wetting  the  inner  surface  of 
the  pail  prevents  contamination  from  it.  The  Exposure  should 
not  be  continued  for  more  than  thirty  seconds.  The  results 
should  be  finally  expressed  in  numbers  of  bacteria  that  would 
fall  into  a  12-inch  pail  under  the  conditions  obtaining  in  the 
trial  during  the  average  time  of  milking  (five  minutes). 

Animals  in  varying  degrees  of  cleanliness  should  be  selected. 
Exposures  should  also  be  made  in  a  similar  manner  under  the 
same  animals  immediately  after  their  flanks  and  udder  have 
been  thoroughly  moistened  with  a  clean  damp  cloth. 

Exercise.  Each  student  will  prepare  four  plates  of  lactose  gelatin 
and  allow  them  to  solidify  :  (1)  expose  two  plates  for  thirty  seconds, 
as  described,  under  animals  which  have  not  been  previously  treated ; 
(2)  expose  the  remaining  two  under  animals  whose  udders  arid  flanks 
have  been  thoroughly  moistened  with  a  clean  damp  cloth. 

Contamination  from  manure.  The  amount  of  contamina- 
tion which  the  milk  receives  from  animals  whose  udders  and 
flanks  are  soiled  with  manure  or  mud  is  not  determined  with 
any  degree  of  accuracy  by  the  exposure  of  plates  in  the  manner 
described,  since  each  particle  of  dirt  may  cause  the  develop- 
ment of  a  colony,  although  it  may  have  carried  hundreds  of 
bacteria  which  would,  in  large  part,  be  scattered  through  the 
milk,  on  account  of  the  solubility  of  the  manure.  To  gain  a 
more  correct  idea  of  the  extent  of  contamination  that  may 
come  from  this  source,  a  quantitative  determination  of  the 
bacteria  in  manure  should  be  made  by  collecting  some,  if  pos- 
sible, from  the  flanks  of  animals.  One  gram  should  be  weighed 
out  on  a  piece  of  sterile  filter  paper.  Place  this  in  a  definite 
amount  of  sterile  water  and  shake  until  the  manure  is  dis- 
integrated and  dissolved  as  completely  as  possible.  Prepare 
plates  with  varying  dilutions,  as  in  the  quantitative  analysis 
of  milk. 


58  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Exercise.  Each  student  will  make  a  quantitative  examination  of 
fresh  manure  and  also  of  dried  manure  removed  from  the  flanks 
of  cows. 

A  piece  of  sterile  filter  paper  4  inches  square  is  placed  on  the  scale 
pan,  and  on  this  a  smaller  piece.  One  gram  of  manure  is  weighed  out 
on  the  sterile  filter  paper  ;  the  upper  paper  and  manure  are  trans- 
ferred to  a  flask  of  sterile  water  (500  cc.).  Make  as  even  a  suspen- 
sion as  possible.  The  dilutions  should  be  very  large,  especially  in 
case  of  the  fresh  manure,  — 1/10,000,  1/100,000,  and  1/1,000,000 
grams,  for  example. 

Plain  agar  medium  should  be  used  and  the  plates  incubated  for 
forty-eight  hours  at  37°  C.,  or  at  20°  C.  for  four  to  five  days. 

Express  the  results  as  the  number  per  gram  of  sample  examined. 

Contamination  from  hair.  Hairs  removed  from  the  flanks 
and  udders  of  even  the  cleanest  animal  bear  more  or  less  dust 
and  bacteria.  The  extent  of  this  contamination  can  be  meas- 
ured by  removing,  with  a  pair  of  scissors,  some  of  the  long 
hairs  from  the  udders  and  flanks  and  placing  them  in  sterile 
Petri  dishes  for  transportation  to  the  laboratory.  Lactose- 
gelatin  plates  are  prepared  as  previously.  When  the  plates 
are  well  solidified  three  or  four  hairs  are  placed  on  the  sur- 
face of  the  medium  by  means  of  a  pair  of  forceps  or  the 
platinum  loop.  The  hair  should  be  pressed  down  so  as  to 
be  thoroughly  in  contact  with  the  medium.  A  separate  cul- 
ture dish  should  be  used  for  the  hairs  from  different  animals. 
The  results  should  be  expressed  as  the  average  number  of 
bacteria  per  linear  inch  on  the  hairs  removed  from  the  vari- 
ous animals. 

Exercise.  Each  student  will  collect  several  individual  hairs  from 
four  animals  showing  varying  degrees  of  cleanliness,  and  determine 
the  number  of  organisms  on  the  same,  noting  the  relative  number 
of  molds,  liquefying  and  nonliquefying  bacteria. 

Contamination  from  interior  of  udder.  Bacteria  may  gain 
entrance  to  the  interior  of  the  udder  and  establish  themselves 


CONTAMINATION  OF  MILK  59 

in  the  secreting  portion  of  the  gland  and  in  the  milk  ducts 
and  cistern.  The  portals  of  entrance  are  two.  When  patho- 
genic organisms  are  present  in  the  circulating  blood,  they 
may  pass  into  the  udder  and  possibly  into  the  milk  ducts 
(foot-and-mouth  disease).  The  more  frequent  manner  is  for 
the  organisms  to  lodge  in  'the  tissues  of  the  udder,  where 
they  grow,  and  from  which  they  may  be  discharged  into  the 
milk  ducts  by  the  breaking  of  the  abscess  (inflammation  of 
the  udder,  or  tuberculosis). 

The  second  portal  of  entrance  is  through  the  exterior 
openings  of  the  teats.  For  a  short  time  after  milking  the 
end  of  the  teat  is  moist,  and  in  the  case  of  animals  that 
leak  the  milk,  constantly  so.  Bacteria  collect  at  this  point 
and  gain  their  way  into  the  teat,  milk  cistern,  and  milk 
ducts.  Most  of  the  forms  that  enter  in  this  way  do  not 
find  favorable  conditions  for  growth,  and  soon  disappear. 
Others  are  able  to  grow  to  some  extent,  and  may  penetrate 
to  the  secreting  tissues  of  the  gland.  As  a  rule,  no  patho- 
logical disturbance  follows  the  entrance  of  the  bacteria,  as 
most  are  saprophytic  forms.  At  times  cases  of  udder  inflam- 
mation are  produced  by  organisms  entering  through  the  teat. 
Contagious  garget  is  spread  by  the  milker  in  this  way. 

In  order  to  determine  the  importance  of  this  source  of 
bacteria,  samples  of  milk  should  be  collected  in  such  a 
manner  as  to  exclude  other  sources  of  contamination.  The 
samples  should  be  collected  at  different  times  during  the 
process  of  milking.  The  results  obtained  will  give  an  idea 
of  the  portion  of  the  gland  in  which  the  largest  number  of 
bacteria  are  found.  If  the  first  few  streams  drawn  from  a 
quarter  contain  many  more  bacteria  than  the  milk  collected 
at  the  end  of  the  milking  process,  then  it  is  evident  that  the 
bacteria  were  largely  in  the  teat  and  milk  cistern. 


60  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

A  sample  of  the  foremilk,  the  first  10-15  cc.  of  a  single 
quarter,  should  be  drawn  directly  into  a  sterile,  wide-mouthed 
bottle  or  flask,  as  well  as  a  sample  of  the  strippings  from  the 
same  quarter.  A  sample  should  also  be  taken  from  the  pail 
after  the  milking  of  the  individual  animal  is  completed. 
Quantitative  analyses  are  to  be  made  in  the  usual  way.  The 
various  samples  should  be  kept  at  room  temperature  and 
frequent  observations  made,  in  order  to  determine  the  keep- 
ing quality  and  the  nature  and  flavor  of  the  curdled  milk. 

Exercise.  Each  student  will  collect  samples  of  foremilk  and  strip- 
pings  from  one  quarter  of  each  of  two  cows,  and  also  from  the  pail 
at  the  end  of  the  milking  of  each  animal,  and  examine  the  samples 
quantitatively. 

Contamination  from  utensils.  The  amount  of  contamina- 
tion which  the  milk  receives  from  the  various  utensils  used 
in  the  handling  depends  entirely  upon  the  cleanliness  of  the 
same.  The  degree  of  cleanliness  of  these  utensils  turns  not 
only  on  the  manner  in  which  they  have  been  washed,  but 
upon  the  construction  and  condition  of  the  utensil.  A  milk 
pail  pressed  from  a  single  sheet  of  metal,  and  well  tinned, 
is  easily  cleaned,  for  it  has  no  crevices  and  joints  into  which 
the  milk  can  penetrate,  and  from  which  it  is  impossible  to 
remove  it.  A  pail  constructed  in  the  ordinary  way,  with 
folded  seams,  is  less  easy  to  keep  in  a  condition  of  bacterio- 
logical cleanliness.  With  the  increased  complexity  of  con- 
struction it  becomes  more  and  more  difficult  to  maintain  any 
utensil  in  a  sanitary  condition. 

On  the  farm  most  of  the  utensils  are  of  such  a  nature  as 
to  be  cleaned  easily,  but  in  the  creamery,  cheese  factory,  etc., 
where  cream  separators,  pasteurizers,  and  numerous  pipes  are 
made  use  of  in  handling  the  milk,  the  difficulty  is  increased. 
On  the  farm  the  most  difficult  utensils  to  keep  clean  are  the 


CONTAMINATION   OF  MILK  61 

farm  separator  and  the  milking  machine,  where  this  is  in  use. 
On  many  farms  the  separators  are  not  taken  apart  for  wash- 
ing after  each  period  of  use,  but  the  apparatus  is  rinsed  out 
with  water,  either  hot  or  cold,  and  left  in  an  assembled  con- 
dition. Sometimes  the  machine  is  taken  apart  and  thor- 
oughly cleaned  only  at  considerable  intervals,  such  as  once  a 
week,  reliance  for  cleanliness  being  placed  on  the  use  of  a  solu- 
tion of  washing  powder  and  subsequent  rinsing  with  water. 

The  long  rubber  tubes  which  are  necessary  with  the  milk- 
ing machine  cannot  be  cleaned  perfectly,  and  must  be  kept 
in  an  antiseptic  liquid  in  order  to  prevent  bacterial  growth. 
For  this  purpose  limewater  or  a  weak  solution  of  formalin 
is  preferably  used,  although  brine  solutions  have  sometimes 
been  recommended. 

The  number  of  bacteria  adhering  to  any  utensil,  will  de- 
pend upon  the  condition  of  the  surface.  If  it  is  smooth,  as 
in  a  new,  well-tinned  pail,  the  number  will  be  much  less 
than  if  the  surface  is  roughened  by  rust.  The  difficulty  in 
rinsing  the  milk  from  even  a  perfectly  smooth  surface  can 
be  illustrated  by  filling  a  pipette  with  whole  milk  and  then 
trying  to  remove  all  visible  traces  of  the  milk  by  passing  a 
stream  of  water  through  it. 

If  the  milk  is  allowed  to  dry  on  a  utensil,  it  is  impossible 
to  remove  it  by  ordinary  means,  since  the  casein  when  dried 
forms  a  glue-like  substance,  which  softenfe  in  water  very 
slowly  and  can  be  removed  only  by  scouring. 

Cloth  strainers  are  likely  to  add  more  bacteria  to  the  milk 
than  they  remove,  unless  they  are  so  treated  as  to  prevent  all 
growth  in  the  cloth.  A  strainer  well  rinsed  in  cold  water, 
then  in  boiling  water,  wrung  dry,  and  hung  in  the  sun,  or 
where  it  will  dry  rapidly,  will  contain  small  numbers  of  bac- 
teria. A  strainer  simply  rinsed  in  cold  or  lukewarm  water 


62  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

and  hung  in  a  damp  milk  room  or  creamery  will  soon  be  a 
source  of  contamination. 

The  number  of  bacteria  to  be  found  in  any  utensil  is  de- 
pendent not  only  on  the  manner  of  washing,  but  on  whether 
or  not  it  has  been  thoroughly  dried.  If  small  amounts  of 
water  are  left  in  cans,  pails,  bottles,  etc.,  a  profuse  growth  of 
bacteria  will  take  place,  for  the  water  will  contain  sufficient 
organic  matter  for  food,  no  matter  how  carefully  the  utensil 
was  cleaned. 

The  bacteriological  condition  of  any  utensil  can  be  deter- 
mined by  rinsing  it  with  sterile  water  and  preparing  quanti- 
tative plates  from  the  water.  The  walls  and  bottoms  of  pails, 
cans,  etc.,  should  be  scrubbed  with  a  sterile  brush,  especial 
attention  being  given  to  the  seams.  Cloth  strainers  may  be 
rinsed  in  sterile  water  and  this  examined.  Milking  machines 
should  have  a  considerable  volume  of  water  passed  through 
the  teat  cups  and  rubber  tubes  into  the  can.  Separators  are 
examined  in  a  similar  way. 

Exercise.  Each  student  will  test  the  bacteriological  condition  of 
pails,  strainers,  and  farm  separators,  which  have  been  treated  in  the 
various  ways  generally  employed  in  farm  and  creamery  practice. 

PAILS  AND  CANS.  Sterilize  two  flasks  of  water  containing  100  cc. 
each.  Place  two  test-tube  brushes  in  large  test  tubes,  stopper  with 
cotton,  and  sterilize  with  the  flask  of  water.  Pour  a  flask  of  the 
sterile  water  into  the  utensil  to  be  examined,  scrub  the  walls  and 
bottom,  and  especially  the  seams  with  the  test-tube  brush.  Return 
the  water  to  the  flask  and  from  it  prepare  quantitative  lactose-agar 
plates.  From  the  data  obtained,  calculate  the  number  of  bacteria  that 
would  have  been  added  to  the  milk  in  case  the  vessel  used  had  been 
filled  with  milk. 

CLOTH  STRAINERS.  Sterilize  a  liter  of  water  in  a  small  pail  or 
can.  Rinse  a  strainer  in  this  water  as  well  as  possible,  wringing  out 
the  cloth.  Examine  the  water  quantitatively. 

FARM  SEPARATORS.  A  quantity  of  milk  should  be  passed  through 
the  separator  and  the  milk  removed  by  flushing  the  machine  with  a 
couple  of  gallons  of  cold  water. 


CONTAMINATION  OF  MILK  63 

From  twelve  to  twenty -four  hours  later  pass  through  the  machine 
a  definite  quantity  of  water  which  has  been  boiled  for  a  few  moments, 
in  order  to  sterilize  it,  and  then  cooled.  Catch  samples  of  the  water  in 
sterile  test  tubes  and  examine  quantitatively. 

Repeat  the  trial  by  passing  milk  through  a  separator  for  four 
days,  cleaning  the  separator  after  each  day's  use  by  flushing  with 
cold  water,  then  with  a  hot  solution  of  washing  powder,  then  with 
hot  water,  to  remove  the  washing  powder.  At  the  end  of  the  period 
pass  sterile  water  through  and  examine  as  before. 

Washing  utensils.  The  student  should  study  the  influ- 
ence which  the  manner  of  washing  utensils  has  on  their 
germ  content. 

Few  farms  are  provided  with  facilities  for  production  of 
steam,  so  it  is  much  more  difficult  to  clean  utensils  properly 
on  the  farm  than  in  the  factory. 

Cans  or  pails  should  be  washed  in  the  same  manner  as  on 
the  farm,  and  the  results  obtained  compared  with  those  ob- 
tained from  the  examination  of  a  pail  or  can  washed  in  a 
similar  way  and  then  steamed. 

Exercise.  Each  student  will  wash  the  cans  furnished  him  in  the 
following  manner  :  Rinse  with  cold  water,  wash  in  a  warm  solu- 
tion of  washing  powder,  and  rinse  with  scalding  water.  One  of  the 
cans  should  be  examined  as  described  in  the  previous  exercise.  The 
other  should  be  steamed  for  thirty  seconds  over  a  steam  jet  and  then 
examined. 

Plan  for  the  production  of  sanitary  milk.  Each  student 
will  prepare  and  submit  for  examination  a  plan  for  the 
establishment  of  a  plant  for  the  production  of  milk  of  high 
grade,  and  for  the  management  of  the  plant.  This  plan 
should  include  everything  which  may  have  a  direct  or 
indirect  relation  to  the  milk  as  human  food.  The  con- 
struction of  the  stable  has  an  indirect  relation  to  the  con- 
tamination of  the  milk,  the  health  of  the  animal  to  the 
hygienic  requirements,  etc. 


CHAPTEK  IV 

MILK  FERMENTATIONS 

In  the  study  of  medical  bacteriology  the  specific  causal 
organism  of  any  disease  is  the  important  thing ;  hence  great 
emphasis  is  laid  on  the  study  of  the  organism  in  question,  — 
its  isolation  and  its  identification.  In  dairy  bacteriology  the 
various  types  of  fermentations  are  not  produced  by  a  single 
species,  but  rather  by  a  group  of  organisms  capable  of  caus- 
ing, in  a  general  way,  similar  chemical  changes.  The  organ- 
isms connected  with  these  changes  may  differ  much  among 
themselves.  The  ordinary  souring  of  milk  may  be  produced 
by  a  host  of  bacteria  having  not  only  dissimilar  morpholog- 
ical characteristics  but  cultural  ones  as  well ;  some  of  them 
are  micrococci,  others  small  bacilli,  and  yet  others  large  rod- 
shaped  forms.  In  dairy  bacteriology  the  character  of  the  fer- 
mentation and  the  nature  of  by-products  formed  in  milk 
are  the  important  things.  Hence  the  more  detailed  study 
of  specific  organisms  is  of  somewhat  less  importance  than 
in  medical  bacteriology. 

It  is,  however,  very  desirable  for  the  student  to  study 
representative  organisms  of  each  group,  even  though  the 
specific  forms  are  not  identified,  for  work  of  this  character 
is  needed  to  teach  the  beginner  the  necessity  of  noting  easily 
overlooked  cultural  differences  that  would  not  be  observed 
except  by  close  comparative  study.  In  the  detailed  study 
of  any  organism  certain  points  in  regard  to  the  morphology, 
cultural  characteristics,  and  biochemical  reactions  must  be 

64 


MILK  FERMENTATIONS  65 

determined.  The  important  questions  are  those  that  admit 
of  a  positive  or  negative  answer.  Those  which  involve  de- 
tailed descriptions  are  of  less  importance  to  the  beginner; 
hence,  with  a  few  exceptions,  not  so  much  emphasis  is  laid 
on  the  cultural  characteristics  as  on  the  morphology  and 
the  biochemistry  of  the  organism. 

Preliminary  cultivation.  Whenever  an  organism  is  to  be 
studied  and  an  attempt  made  to  determine  to  what  partic- 
ular group  it  belongs,  it  is  important  that  it  be  in  a  state  of 
high  vitality,  as  otherwise  uniform  results  cannot  be  obtained. 
In  nearly  all  cases  the  optimum  conditions  for  the  growth 
of  an  organism  are  not  known.  It  has  been  determined  that 
by  a  preliminary  cultivation  the  constancy  of  the  results 
obtained  may  be  materially  increased.  The  standard  proce- 
dure of  preliminary  cultivation  is  as  follows :  From  an  agar 
culture  of  the  organism  a  broth  culture  is  made,  and  incu- 
bated for  twenty-four  hours  at  20°  C.  Transfer  from  this 
broth  culture  to  a  second  tube  of  broth,  and  incubate  for 
twenty-four  hours  at  20°  C. ;  repeat  a  third  time.  In  order 
to  be  certain  that  the  final  trials  are  made  with  a  pure 
culture,  from  the  third  broth  tube  gelatin  plates  are  pre- 
pared. From  one  of  the  colonies  on  the  gelatin  plates  in- 
oculate a  tube  of  sloped  agar  and  incubate  for  forty-eight 
hours  at  20°  C.  From  this  the  subsequent  inoculations  are 
to  be  made  into  various  media. 

This  method  of  restoring  the  vitality  of  the  organism  pre- 
supposes that  it  will  find  favorable  conditions  for  growth  on 
the  ordinary  nutrient  media  and  at  a  temperature  of  20°  C. 
With  some  groups  of  bacteria,  important,  in  the  dairy,  this 
method  of  preliminary  cultivation  should  be  modified.  The 
great  group  of  lactic-acid  bacteria  grow  very  poorly,  often 
not  at  all,  in  the  ordinary  nutrient  broth.  If,  however,  this 


66 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


be  qualified  by  the  addition  of  a  fermentable  sugar,  the  or- 
ganisms find  much  more  favorable  conditions  for  growth. 

Primary  tests  for  identification  of  species.  The  primary 
tests  for  the  identification  of  bacterial  species  are  those  that 
can  be  answered  positively  or  negatively.  In  the  study  of  an 
organism  the  following  scheme  should  be  employed :  The  posi- 
tive results  should  be  expressed  by  a  cross  (+),  a  double  cross 
($)  indicating  an  emphatically  positive  result ;  zero  (0)  indi- 
cates a  negative  result ;  a  question  mark  (?),  a  doubtful  result ; 
and  a  blank  space  indicates  that  no  determination  was  made. 


1.  Source 

2.  Number  or  name 


PRIMARY  TESTS  FOR  IDENTIFICATION 
BACTERIAL  SPECIES 


OF 


f  Bacillus 

Form     .     .     . 

.  4  Coccus 

[_  Spirillum 

Size 

Diameter  greater  than  1  /x 

3.  Morphology  . 

Arrangement  . 

J  United  in  filaments 
L  United  in  pairs 

Movement 

Motile 

Structure  . 

J  Flagella  observed 
'  \  Spores 

f  Stains  easily  with  watery  dyes 
Staining  reaction  4  _,    .      .         "       , 
I  Stains  by  Gram  s  method 

• 

r  Turbidity 

Broth  tube     . 

J  Sediment 
I  Pellicle 

Gelatin  plate 

Peculiarities  of  colonies 

4.   Cultural 

f  Surface  growth 

characteristics 

Gelatin  tube 

.  J  Needle  growth 

[^  Liquefaction 

f  Dull  growth 

Agar  tube 

1  Wrinkled  growth 
1  Chromogenesis 

t 

I  Shining 

.MILK  FERMENTATIONS 


6T 


Milk     .     .     . 

Dextrose  broth 
Lactose  broth 

Saccharose  broth 

Nitrate  broth 
Peptone  broth     . 

Relation  to 
oxygen 


f  Coagulation 
.  -j  Degree  of  reaction 
[  Casein  liquefaction 

/Gas 

'  t  Acid 

|  Gas 

Ft  Acid 
i.  Biochemical     Saccharose  broth  |  A"d 
reactions 
Nitrite  formation 

Indol  production 

f  Aerobic 

I\  Facultative  anaerobic 
oxygen        .     .  i 
[_  Anaerobic 
Relation  to  tern-    f  Growth  at  20°  C. 
perature      .     .  \  Growth  at  37°  C. 
Thermal  death  point  below  80°  C. 

The  morphological  study  should  be  made  on  fully  devel- 
oped cultures.  Cultures  which  are  growing  rapidly  may  pre- 
sent a  large  number  of  immature  forms.  Old  cultures  may 
.contain  degenerated  and  altered  forms  (involution  types).  No 
definite  statement  can  be  made  in  regard  to  the  age  of  a  cul- 
ture, but,  as  a  rule,  those  grown  at  37°  C.  should  not  be 
over  twenty-four  to  forty-eight  hours  old,  and  those  grown 
at  20°  C.  from  forty-eight  to  seventy-two  hours  old.  Exam- 
inations of  material  should  be  made  from  both  solid  and 
liquid  media  in  both  stained  and  unstained  preparations. 

In  Appendix  B  is  given  the  scheme  for  the  detailed  study 
of  an  organism,  recommended  by  the  Society  of  American  Bac- 
teriologists, together  with  a  glossary  of  the  terms  employed 
in  describing  the  cultural  characteristics  of  an  organism. 

Action  on  carbohydrates.  The  ability  of  the  bacteria  to 
ferment  sugar  with  the  production  of  gas  and  acid  is  best 
determined  by  the  use  of  the  fermentation  tube,  since  this 


68 


EXPERIMENTAL   DAIRY  BACTERIOLOGY 


procedure  allows  not  only  the  qualitative  determination  to 
be  made,  but  also  permits  of  a  rough  quantitative  analysis 
and  the  determination  of  the  relative  amounts  of  the  com- 
ponent gases  formed. 

In  case  the  fermentation  tubes  filled  with  sugar  broth  have 
not  been  recently  sterilized,  heat  in  the  sterilizer  in  order  to 


FIG.  13.    FROST'S  GASOMETER 

The  amount  of  gas  is  expressed  in  per  cent  of  the  length  of  the  closed  arm  of 
the  fermentation  tube.    (After  Heiuemann) 

drive  off  dissolved  oxygen.  If  they  contain  bubbles  of  gas  on 
being  taken  from  the  sterilizer,  remove  them  by  decantation. . 
When  ready  to  use,  the  liquid  should  fill  the  closed  arm  and 
stand  at  a  low  level  in  the  open  bulb.  When  cool,  inoculate. 
At  frequent  intervals  during  the  incubation,  which,  as  a 
rule,  should  be  made  at  37°  C.,  the  presence  or  absence  of 


MILK   FERMENTATIONS  69 

gas  in  the  closed  arm  should  be  noted,  and  the  amount  of  gas 
determined  by  measuring  the  same  with  Frost's  gasometer. 

When  the  culture  ceases  to  form  gas,  remove  from  the  in- 
cubator, cool  to  room  temperature,  and  measure.  Test  the 
reaction  of  the  liquid  in  the  open  arm  by  the  use  of  litmus 
paper,  or  titrate  a  definite  amount  as  in  the  case  of  milk. 
The  culture  should  not  be  allowed  to  stand  too  long  after 
gas  evolution  ceases,  as  the  carbon  dioxide  formed  is  rapidly 
absorbed  by  the  liquid. 

To  determine  the  relative  proportion  of  the  component 
gases  formed,  fill  the  bulb  of  the  fermentation  tube  with  a 
2  per  cent  solution  of  sodium  hydroxide,  close  the  opening 
of  the  tube  with  the  thumb,  tip  the  tube  so  as  to  allow  the 
gas  to  fill  the  bulb  and  come  in  contact  with  the  alkaline  solu- 
tion ;  return  the  gas  to  the  closed  arm,  and  repeat  the  process 
several  times,  keeping  the  open  arm  tightly  closed.  With  the 
gas  in  the  closed  arm,  remove  the  thumb.  The  liquid  will  rise 
in  the  closed  arm  on  account  of  the  absorption  of  the  C02  by 
the  alkali.  Measure  the  residue,  which  is  usually  considered 
as  hydrogen,  close  the  tube  again  with  the  thumb,  and  bring 
the  gas  into  the  bulb ;  remove  the  thumb  and  introduce  a 
lighted  match.  The  air  mixed  with  the  hydrogen  forms  an 
explosive  mixture.  The  composition  of  the  gas  is  expressed 
by  the  ratio  of  hydrogen  to  carbon  dioxide  (H/C02). 

With  bacteria  that  do  not  grow  at  37°  C.  the  test  should 
be  made  at  20°  C.,  the  time  of  incubation  being  extended  at 
least  four  days. 

A  qualitative  test  for  gas  production  from  the  various 
sugars  can  be  made  by  the  use  of  "shake. cultures."  A  tube 
of  melted  gelatin  or  agar  containing  2  per  cent  of  the  sugar 
to  be  tested  is  inoculated  with  the  culture,  mixed  well,  and 
solidified  by  placing  in  ice  water.  If  the  sugar  is  fermented 


70  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

by  the  organism  with  the  production  of  gas,  the  medium  will 
be  rent  by  the  gas.  Tubes  of  broth  can  also  be  used  for  the 
qualitative  determination  of  gas  by  placing  in  the  tube  before 
sterilization  a  small  piece  of  glass  tubing  sealed  at  the  upper 
end.  In  the  sterilization  the  air  is  completely  driven  out  and 
the  small  tube  filled  with  the  broth.  If  gas  is  formed  in  the 
developing  culture,  bubbles  will  collect  in  the  small  tube. 

When  it  is  desired  to  determine  whether  or  not  an  organ- 
ism is  able  to  ferment  a  certain  sugar,  it  is  essential  that  the 
medium  to  which  the  sugar  is  added  be  free  from  sugar.  In 
media  prepared  from  meat  or  beef  extract,  a  small  amount  of 
muscle  sugar  (inosite)  is  present.  This  can  be  removed  by 
inoculating  the  meat  infusion,  after  straining,  with  a  culture 
of  an  organism  able  to  ferment  inosite  (B.  coli  commuriis),  and 
incubating  for  twelve  to  sixteen  hours,  or  until  all  the  sugar 
is  decomposed.  The  medium  is  then  finished  in  the  usual 
manner. 

In  the  testing  of  sugars  easily  affected  by  heat,  it  is  essen- 
tial that  the  sterilization  be  as  short  as  possible,  otherwise 
the  sugar  may  be  decomposed  into  its  constituent  sugars  and 
these  be  then  fermented.  Saccharose  in  slightly  acid  solu- 
tions is  easily  inverted,  lactose  far  less  easily. 

Exercise.  Inoculate  fermentation  tubes  filled  with  lactose  brotli 
from  the  cultures  furnished.  Determine  the  amount  and  composition 
of  the  gas  produced  (see  p.  69).  Also  inoculate  tubes  of  melted  lac- 
tose agar  from  the  various  cultures. 

Action  on  nitrates.  Many  organisms  have  the  power  of 
reducing  nitrates  with  the  formation  of  nitrites,  and  some- 
times ammonia  and  free  nitrogen.  Nitrate  broth  is  prepared 
by  dissolving  1  gram  of  peptone  in  1  liter  of  tap  water  and 
adding  0.2  grams  of  nitrite-free  potassium  nitrate.  Tube 
and  sterilize  in  the  usual  way.  Inoculate  and  incubate  for 


MILK   FERMENTATIONS  71 

four  days  at  37°  C.,  together  with  an  uninoeulated  tube  to 
serve  as  a  control. 

Test  for  nitrites.  Dissolve  8  grams  of  the  purest  sulpha- 
nilic  acid  in  1000  cc.  of  30  per  cent  acetic  acid  (sp.  gr.  1.041). 
Dissolve  5  grams  of  a-amidonaphthylamine  in  1000  cc.  of  30 
per  cent  acetic  acid ;  filter  through  a  plug  of  washed  absorbent 
cotton.  At  the  end  of  the  period  of  incubation  in  the  nitrate 
broth  remove  3  cc.  of  the  culture  to  a  clean  test  tube  and 
add  2  drops  each  of  the  naphthylamine  and  sulphanilic  acid 
solutions.  The  development  of  a  red  color  indicates  the  pres- 
ence of  nitrites,  the  intensity  of  the  shade  indicating  in  a 
general  way  the  amount  of  nitrites  present.  The  control  tube 
should  be  treated  in  the  same  manner  as  the  culture. 

Test  for  ammonia.  Nessler  solution.  Dissolve  50  grams 
of  potassium  iodide  in  a  minimum  quantity  of  cold  water. 
Add  a  saturated  solution  of  mercuric  chloride  until  a  slight 
but  permanent  precipitate  persists.  Add  400  cc.  of  a  50  per 
cent  solution  of  potassium  hydrate  made  by  dissolving  the 
potassium  hydrate  and  allowing  it  to  clarify  before  using. 
Dilute  to  1  liter  and  allow  the  mixture  to  settle.  One  half 
of  the  remaining  portion  of  the  culture  used  for  the  nitrite 
test  is  removed  to  a  test  tube  and  a  couple  of  drops  of  the 
Nessler  solution  added.  The  presence  of  ammonia  is  indi- 
cated by  a  yellow  color,  or  by  a  yellow  precipitate  when  the 
ammonia  is  present  in  considerable  quantities. 

The  presence  of  ammonia  in  broth  culture  may  be  de- 
tected by  moistening  a  strip  of  filter  paper  with  the  Nessler 
solution  and  placing  the  same  in  the  neck  of  the  tube,  a  yel- 
low to  reddish-brown  color  indicating  the  presence  of  ammonia. 

Exercise.  From  the  cultures  furnished,  inoculate  tubes  of  nitrate 
solution.  Determine,  as  described,  the  ability  of  the  organisms  to 
produce  nitrites  and  ammonia  from  the  nitrate  present. 


72  EXPERIMENTAL   DAIRY  BACTERIOLOGY 

Formation  of  indol.  Many  bacteria  produce  a  number  of 
aromatic  compounds,  the  most  important  of  which  is  indol, 
one  of  the  series  associated  with  nitrogenous  decomposition, 
as  in  fecal  matter.  Indol  is  produced  only  in  the  absence  of 
sugar.  Sugar-free  broth  must  thus  be  used.  The  incubation 
of  the  meat  infusion  must  be  sufficiently  prolonged  to  re- 
move the  sugar,  but  not  so  extended  as  to  permit  of  indol 
formation.  The  use  of  peptone  solution  is  preferable  for  this 
purpose;  to  make  this  solution,  10  grams  of  peptone  are  dis- 
solved in  1000  cc.  of  water,  tubed,  and  sterilized. 

The  broth  is  inoculated  and  incubated  for  four  days  at 
37°  C.  To  test  for  indol  add  1  cc.  of  0.01  per  cent  solution 
of  sodium  nitrite,  mix,  add  a  few  drops  of  concentrated  sul- 
phuric acid,  allowing  it  to  flow  down  the  side  of  the  tube, 
forming  a  layer  at  the  bottom.  If  indol  is  present,  a  pink  or 
red  ring  forms  at  the  junction  of  the  two  liquids,  due  to  the 
formation  of  nitroso-indol.  The  tubes  should  be  allowed  to 
stand  for  half  an  hour  before  final  judgment  is  recorded. 

Exercise.  Inoculate  tubes  of  sugar-free  broth  from  the  cultures 
furnished.  Test  for  indol  as  described. 

Relation  to  oxygen.  The  relation  of  the  bacteria  to  oxygen 
may  be  determined  by  noting  the  character  of  the  growth  in 
the  fermentation  tube.  If  the  tubes  are  heated  in  the  steril- 
izer shortly  before  inoculation,  the  oxygen  dissolved  in  the 
medium  will  be  driven  off,  and  in  the  closed  arm  anaerobic 
conditions  will  prevail.  If  the  developing  growth  is  limited 
to  the  open  arm  of  the  tube,  the  organism  is  an  obligate 
aerobe ;  if  confined  to  the  closed  arm,  it  is  an  obligate  an- 
aerobe ;  if,  however,  the  growth  is  present  in  both  open  and 
closed  portions  of  the  tube,  it  possesses  the  faculty  of  growing 
under  either  condition,  and  is  called  a  facultative  organism. 


MILK   FERMENTATIONS 


73 


More  exact  results  can  be  obtained  by  the  use  of  special 
anaerobic  culture  methods  (see  p.  82). 

Exercise.  Note  the  presence  or  absence  of  growth  in  the  closed 
and  open  arms  of  the  fermentation  tubes  inoculated  with  various 
cultures  (p.  70). 


ABC 
FIG.  14.    TYPES  OF  GROWTH  IN  FERMENTATION  TITBES 

A,  facultative  anaerobic  organism ;  H,  aerobic  organism ;  6',  gas  formation  by 
facultative  anaerobic  organism.   (After  Smith) 

Relation  to  temperature.  The  relation  of  the  developing 
organism  to  temperature  is  determined  by  noting  the  com- 
parative rapidity  of  growth  at  20°  C.  and  at  37°  C.  Every 
organism  has  a  more  or  less  definite  temperature  range  from 
the  minimum  to  the  optimum,  and  finally  to  the  maximum, 
temperature  at  which  growth  will  go  on.  Generally  most 
bacteria  prefer  a  relatively  high  temperature,  such  as  blood 
heat,  but  some  of  the  fermentative  types  find  ordinary  air 
temperatures  almost  as  suitable  as  the  higher  degree  of  heat. 

The  ability  of  the  organism  to  resist  the  temperature  of 
80°  C.  for  fifteen  minutes  should  be  determined,  since  it  gives 


74  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

indications  as  to  whether  the  organism  produces  spores  or 
not.  A  broth  culture  forty-eight  hours  old,  grown  at  37°  C., 
is  used.  In  a  water  bath  heated  to  80°  C.  place  four  tubes  of 
nutrient  broth  in  immediate  contact  with  the  thermometer. 
After  fifteen  minutes'  exposure  to  this  temperature  inoculate 
each  tube,  without  removing  from  the  water,  with  three  loop- 
fuls  of  the  broth  culture,  using  care  not  to  contaminate  the 
glass  above  the  surface  of  the  liquid.  Continue  the  exposure 
for  fifteen  minutes,  remove  the  tubes,  and  cool  at  once.  Incu- 
bate for  at  least  seven  days.  Organisms  producing  true  endo- 
spores  are  able  to  withstand  this  temperature.  These  are  the 
types  that  are  so  difficult  to  destroy  in  the  sterilization  of 
milk.  Organisms  resisting  this  temperature  are  classed  as 
spore  forming. 

For  the  more  detailed  determination  of  the  thermal  death 
point  of  the  organism  the  student  is  referred  to  standard 
text-books  on  general  technique. 

Exercise.  Determine  the  optimum  temperature  for  growth  of  the 
organisms  furnished,  and  also  whether  or  not  they  produce  spores. 

Chromogenesis.  The  formation  of  pigment  is  usually  most 
apparent  on  agar  cultures.  Cultures  should  be  incubated  at 
both  20°  and  37°  C.  Some  organisms  produce  pigment  only 
under  special  conditions,  as  B.  mesentericus  ruler  when  grow- 
ing on  potato.  Free  oxygen  is  an  essential  with  practically  all 
types.  Therefore  the  cultures  must  be  under  obligate  aerobic 
conditions. 

Action  on  milk.  The  action  of  the  various  organisms  on 
milk  is  of  the  very  greatest  importance  to  the  dairy  bacteri- 
ologist, and  especial  attention  should  be  paid  to  the  determi- 
nation of  the  points  which  are  enumerated  on  the  following 
page. 


MILK  FP:RMENTATIONS  75 

1.  Presence  of  curd. 

2.  Time  required  to  curdle. 

3.  Character  of  curd,  —  acid  or  rennet,  homogeneous  or 

showing  gas  holes,  hard  or  soft. 

4.  Whey,  —  amount,  clear  or  turbid,  reaction. 

5.  Digestion  of  curd,  —  partial,  complete. 

6.  Digestion  without  previous  curdling. 

7.  Odor. 

8.  Gas. 

9.  Change  produced  by  boiling  a  few  seconds. 
10.  Amount  of  acid  produced. 

Plain  milk  may  be  used  or  that  modified  with  litmus. 
The  reaction  of  milk  or  whey  may  be  tested  by  removing  a 
loopful  and  placing  on  delicate  litmus  paper.  Care  must  be 
exercised  sometimes  in  discriminating  between  a  rennet  and 
an  acid  curd.  Reliance  cannot  be  placed  on  the  chemical  re- 
action, as  a  milk  curdled  with  a  rennet-like  enzyme  is  gen- 
erally acid  to  some  extent.  The  digestion  of  casein  cannot 
always  be  determined  with  certainty  in  tube  cultures.  The 
curd  may  sometimes  shrink  in  volume  and  cause  extrusion 
of  the  whey  without  \mdergoing  digestive  changes.  In  doubt- 
ful cases  digestion  can  be  definitely  determined  by  the  use  of 
milk  agar,  which  is  prepared  as  follows :  A  tube  of  nutrient 
agar  is  melted  and  allowed  to  cool  to  50°-55°  C.  With  a 
sterile  pipette  10-15  per  cent  of  sterile  skwn  milk  is  added 
to  it,  the  mixture  being  poured  into  a  Petri  dish  and  allowed 
to  solidify.  The  plate  is  inoculated  with  the  culture  to  be 
tested  by  making  a  streak  culture  with  the  loop.  The  action 
of  the  organism  on  the  casein  is  thus  localized,  and  can  be 
noted  even  though  it  is  very  slight.  The  diffused  proteid 
(casein)  is  peptonized  or  rendered  soluble  by  the  action  of 
digestive  ferments.  The  action  of  acid-producing  organisms 


76  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

when  the  media  contains  salt  (NaCl)  simulates  closely  that 
of  true  digestion,  but  can  be  differentiated  from  it  by  flooding 
the  plates  with  dilute  HC1  (1  per  cent),  when,  in  case  the 
change  has  been  due  to  true  digestion,  the  clear  zone  about 
the  culture  will  remain.  If  the  clearing  was  caused  by  the 
solvent  effect  of  the  acid,  the  addition  of  a  larger  quantity  of 
acid  will  cause  a  reprecipitation. 

The  presence  of  gas  is  easily  noted  in  the  case  of  organisms 
producing  a  curd.  In  other  types  where  no  curd  is  formed, 
as,  for  example,  in  the  case  of  lactose-fermenting  yeasts,  the 
presence  of  gas  may  be  indicated  by  gently  shaking  the  tube 
and  noting  the  evolution  of  bubbles. 

Organisms  of  the  same  species  vary  greatly  in  the  amount 
of  acid  produced.  The  amount  of  acid  can  be  determined  in 
tube  cultures  by  adding  to  a  tube  of  milk  5  cc.  of  distilled 
water.  Mix  well,  turn  into  a  graduated  cylinder,  note  the 
volume,  transfer  the  mixture  to  an  evaporating  dish  or  an 
Erlenmeyer  flask,  add  a  few  drops  of  the  phenolphthalein 
indicator,  and  titrate  with  N/20  NaOH.  Express  the  results 
as  per  cent  of  lactic  acid. 

.      . ,      cc.  of  N/20  x  0.45 
Per  cent  of  acid  ==  -  —  • 

cc.  of  milk 

In  studying  the  various  organisms  illustrating  the  different 
kinds  of  fermentations  four  tubes  of  milk  should  always  be 
inoculated.  Two  of  these  should  be  incubated  at  20°  C.  and 
the  remaining  two  at  37°  C. 

Acid  fermentation.  The  normal  souring  or  acid  fermenta- 
tion in  milk  is  usually  caused  by  a  number  of  different  kinds 
of  bacteria  growing  together.  In  one  sample  of  milk  one  type 
may  predominate ;  in  a  second  sample  a  different  type  may 
be  most  active ;  in  a  third  still  another  form  may  be  present 
in  greatest  numbers. 


MILK    FKKMKXTATIOXS  77 

Nearly  all  of  the  acid-forming  bacteria  which  are  of  prac- 
tical importance  may  be  placed  in  two  great  groups:  (1)  the 
group  including  the  bacteria  which  are  desirable,  especially 
from  the  standpoint  of  the  butter  and  cheese  maker,  B.  lactis 
acidi  Leichrnann  (Streptococcus  lacticus  Kruse)  being  the  most 
important  representative  of  this  group ;  (2)  the  group  includ- 
ing the  bacteria  which  are  undesirable  in  milk,  B.  coli  corn- 
munis  and  the  related  form  B.  lactis  aerogenes,  both  appearing 
in  a  number  of  varieties,  being  the  important  representatives. 
A  detailed  study  should  be  made  of  pure  cultures  of  these 
organisms.  In  this  study  attention  should  be  directed  to  the 
changes  produced  in  milk,  especially  to  .the  nature  of  the  curd 
formed,  the  odor  and  taste  of  the  milk,  and  the  rate  and 
quantity  of  acid  produced  at  20°  C.  and  at  37°  C. 

The  student  should  also  collect  samples  of  milk,  allow  them 
to  sour,  and  judge  by  the  nature  of  the  curd  produced  the  type 
of  organisms  concerned  in  the  souring.  From  the  samples  of 
milk  showing  different  types  of  curd,  plates  should  be  prepared 
with  lactose  agar  in  order  to  isolate  the  organisms.  Kepre- 
sentatives  of  the  various  groups  should  be  studied  in  detail. 

Determination  of  the  group  to  which  the  organism  be- 
longs. The  grouping  may  best  be  made  by  the  inoculation 
of  litmus-milk  tubes  from  the  individual  colonies.  The  re- 
sults thus  obtained  should  show  whether  the  organism  is 
to  be  included  in  either  of  the  groups  mentioned  above,  or 
belongs  to  some  other. 

Exercise.  Each  student  will  make  a  detailed  study  of  the  cultures 
furnished  in  accordance  with  the  outline  given  on  p.  66.  Collect 
samples  of  milk,  and  from  them  isolate  representatives  of  the  two 
groups  of  lactic  bacteria  and  study  in  detail. 

Digesting  and  sweet-curdling  fermentation.  These  changes 
are  produced  by  various  kinds  of  bacteria.  Many  of  the  coccus 


78  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

forms  present  in  fresh  milk  liquefy  casein.  Spore-bearing  ba- 
cilli are  frequently  the  cause  of  these  fermentations.  The  most 
important  representatives  of  the  group  are  the  spore-produc- 
ing organisms  persisting  in  milk  after  heating.  Various  kinds, 
as  shown  by  their  action  in  litmus  milk,  should  be  isolated 
and  studied. 

Exercise.  Each  student  will  procure  samples  of  milk  and  heat 
them  to  100°  C.  for  twenty  or  thirty  minutes.  Incubate  for  six  to 
eight  days  at  20°  C.  From  the  samples  showing  curdling  and  a  neu- 
tral or  alkaline  reaction,  isolate  the  causal  organisms  by  means  of 
gelatin  plates.  Study  in  detail. 

Alcoholic  fermentation.  The  production  of  alcohol  is  al- 
most exclusively  limited  to  the  action  of  yeasts.  Some  bac- 
teria produce  alcohol,  but  in  such  small  quantities  that  it  can 
be  detected  only  by  delicate  chemical  tests.  The  ordinary 
yeasts  —  beer,  wine,  and  bread  yeasts  —  cannot  ferment  milk 
sugar,  and  hence  are  of  little  importance  in  the  dairy.  Yeasts 
that  are  able  to  ferment  milk  sugar  are  important  in  the 
production  of  fermented  drinks  from  milk,  kefir,  koumiss, 
leben,  matzoon,  etc.  Lactose-fermenting  yeasts  are  widely  dis- 
tributed in  milk,  butter,  and  cheese,  and  especially  in  whey 
from  vats  in  which  a  part  of  the  whey  is  allowed  to  remain 
from  day  to  day. 

In  order  to  isolate  the  yeasts  from  a  sample  of  milk,  etc., 
an  acid  medium  is  used.  The  acidity  of  the  medium  may  be 
increased  to  such  a  point  as  to  'exclude  all  bacterial  growth 
and  still  allow  yeasts  to  grow.  This  is  accomplished  by 
adding  to  the  melted  lactose  agar,  just  before  plating,  about 
1  per  cent  of  tartaric  acid,  in  the  form  of  a  30  per  cent 
solution.  The  acid  cannot  be  added  to  the  agar  before  ster- 
ilization as  the  solidifying  properties  of  the  agar  would  be 
destroyed. 


MILK   FERMENTATIONS  79 

In  order  to  determine  whether  the  various  types  of  colonies 
appearing  on  the  plates  are  able  to  ferment  lactose,  tubes  of 
sterile  milk  or  whey  are  inoculated,  incubated  at  20°  C.  and 
at  37°  C.  The  production  of  gas  (C02),  which  becomes  evi- 
dent on  shaking  the  tube  slightly,  is  evidence  of  the  fermen- 
tation of  the  lactose.  Few  of  the  yeasts  produce  any  curdling 
of  the  milk.  The  presence  of  alcohol  is  usually  made  evident 
by  the  odor.  The  iodoform  test  for  alcohol  should  be  made 
by  distilling  over  a  small  quantity  from  a  mass  culture  in 
milk  or  whey,  made  alkaline  with  milk  of  lime.  Add  to  5  cc. 
of  the  distillate  three  or  four  drops  of  a  10  per  cent  solution  of 
iodine  in  potassium  iodide,  until  a  permanent  reddish-brown 
color  is  obtained.  Decolorize  by  adding  10  per  cent  KOH 
drop  by  drop.  Allow  the  tube  to  stand  in  warm  water  until 
a  sediment  collects.  Examine  with  the  £-inch  lens  for  the 
characteristic  crystals  of  iodoform.  The  method  should  first 
be  tried  with  distilled  water  to  which  a  few  drops  of  alcohol 
have  been  added. 

Ropy  fermentation.  The  peculiar  change  in  milk,  known 
as  ropy,  slimy,  or  stringy  milk,  may  be  produced  by  a  large 
number  of  forms  of  bacteria.  With  many  it  is  apparently  a 
normal  property,  with  others  it  is  an  evidence  of  degeneration 
changes.  Some  types  of  lactic-acid  bacteria,  on  cultivation, 
cease  to  curdle  the  milk  in  a  normal  manner,  but  produce 
in  it  varying  amounts  of  a  slimy  material. ' 

The  upper  layers  of  cream  and  milk  in  bottles  may  become 
slimy,  due  to  the  growth  of  aerobic  organisms  (B.  mesenteri- 
cns  vulgatus).  The  slime-producing  organisms  proper  may 
or  may  not  produce  acid.  The  organisms  studied  by  Ward, 
Harrison,  etc.,  belong  to  the  latter  group  (B.  lactis  viscosus). 
The  bacteria  present  in  the  slimy  whey  (lange  wei)  of 
Holland  (Streptococcus  ffollandicus),  in  the  taettemoelk  of 


80  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Norway    (B.   lactis   longi    Troili-Petersson),    belong    to   the 
acid-producing  group. 

In  the  study  of  this  type  of  fermentation  especial  atten- 
tion should  be  devoted  to  the  cause  of  the  ropy  condition.  It 
may  be  due  to  the  presence  of  a  greatly  thickened  cell  wall 
(capsule),  which  may  be  detected  by  appropriate  staining 
(p.  48),  or  to  the  presence  of  a  mucus-like  substance  in  the 
milk  (galactan). 

Exercise.  Each  student  will  make  a  detailed  study  of  the  organ- 
isms furnished,  B.  lactis  lonf/i. 

Bitter  fermentation.  A  bitter  taste  in  milk  may  be  pro- 
duced by  a  large  variety  of  bacteria.  Many  of  the  digesting 
bacteria  produce  a  bitter  taste  in  milk,  due  to  the  formation 
of  peptones  from  the  albumen  and  casein.  Nonliquefying 
acid-forming  bacteria  may  produce  a  specific  bitter  principle 
in  milk,  and  the  same  may  be  formed  by  certain  types  of 
liquefying  acid-producing  bacteria. 

Exercise.  The  various  milk  cultures  studied  in  the  digesting  and 
sweet-curdling  fermentations  should  be  tested  for  bitterness. 

Cycle  of  fermentations  in  milk.  Under  natural  conditions 
the  decomposition  of  organic  matter  is  accomplished  not  by 
a  single  form  of  life,  but  by  a  greater  or  less  variety  of  organ- 
isms growing  together  (symbiosis) ;  or  one  form  prepares  the 
way  for  another,  through  the  establishment  of  a  favorable 
environment,  as  in  the  preparation  of  a  suitable  food  or  a 
favorable  reaction  in  the  medium  (metabiosis).  Milk  is  no 
exception  to  the  rule.  During  the  act  of  withdrawal  from 
the  animal  it  becomes  contaminated  with  certain  classes  of 
bacteria  and  molds,  which  make  themselves  manifest  in  a 
constant  and  regular  sequence  in  ordinary  milks.  On  ac- 
count of  their  small  numbers  it  may  be  impossible  to  find  in 


MILK   FERMENTATIONS  81 

the  fresh  milk  the  various  types  of  bacteria  and  molds  con- 
cerned in  the  changes  which  the  milk  will  undergo.  For 
instance,  it  is  frequently  impossible  to  demonstrate  by  plate 
cultures  the  presence  of  acid-forming  bacteria  or  of  acid- 
destroying  molds  in  fresh  milk.  If,  however,  the  milk  is 
allowed  to  remain  for  some  days  at  ordinary  temperatures, 
these  forms  nearly  always  appear. 

Exercise.  In  each  of  two  liter  flasks  should  be  placed  raw  skim 
milk  to  the  depth  of  1  inch,  the  flasks  being  closed  with  cotton 
plugs.  One  of  the  flasks  should  then  be  heated  to  75°  C.  for  a  few 
moments.  Incubate  both  flasks  at  20°  C.  for  at  least  a  month,  not- 
ing at  frequent  intervals  the  changes  occurring. 

From  the  white  velvet-like  growth  appearing  on  the  raw  milk 
(Oidium  lactis)  microscopic  preparations,  stained  and  unstained,  should 
be  prepared. 

Explain  the  various  changes  noted  in  the  two  flasks. 

Butyric-acid  fermentation.  In  the  soil,  in  manure,  and  in 
barn  dust  are  constantly  found  bacteria  that  are  able  to  fer- 
ment various  carbohydrates,  such  as  dextrose  and  lactose, 
with  the  production  of  butyric  acid  and  other  by-products, 
such  as  lactic  and  acetic  acids,  carbon  dioxide,  and  hydrogen. 

Milk  usually  contains  representatives  of  this  group  of  bac- 
teria. Butter  and  cheese  also  contain  them,  and  are  some- 
times injured  by  them. 

As  a  group  the  butyric-acid  bacteria  are  characterized  by 
their  relation  to  oxygen.  They  are  anaerobic,  and  grow  only 
in  the  absence  of  free  oxygen  when  in  pure  culture.  In  milk, 
butter,  and  cheese  the  oxygen  is  consumed  by  the  aerobic 
forms  present ;  hence  the  conditions,  as  far  as  oxygen  is  con- 
cerned, are  favorable  for  the  butyric-acid  bacteria.  Other  con- 
ditions are  less  favorable.  The  large  amount  of  acid  formed 
in  milk  and  cheese  by  the  lactic-acid  bacteria  inhibits  the 
butyric-acid  bacteria. 


82  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

In  butter  almost  no  sugar  is  present.  Without  it  the 
growth  of  the  butyric-acid  bacteria  cannot  occur. 

The  butyric-acid  bacteria  form  very  resistant  spores.  Milk 
that  has  been  heated  will  frequently  undergo  an  acid  fermen- 
tation, especially  when  incubated  at  37°C.  Gas  is  usually 
abundant.  This  acid  fermentation  of  heated  milk  is  due  to 
the  spores  of  the  butyric-acid  bacteria  that  have  not  been 
killed  by  the  heating.  Such  milk  usually  has  a  very  offen- 
sive odor. 

In  order  to  isolate  the  organisms  in  pure  culture  some 
one  of  the  many  ways  suggested  for  the  removal  of  the 
free  oxygen  from  the  culture  vessel  must  be  employed.  The 
oxygen  can  be  absorbed  by  an  alkaline  solution  of  pyrogallic 
acid,  or  the  air  may  be  replaced  by  an  inert  gas  such  as  hy- 
drogen. A  combination  of  the  two  methods  is  preferable. 
By  exhausting  the  air  from  the  vessel  by  means  of  a  water 
pump,  and  then  filling  the  vessel  with  hydrogen  and  again 
exhausting,  the  time  required  to  remove  the  oxygen  may  be 
shortened. 

The  apparatus  illustrated  in  Fig.  15  will  be  found  very  con- 
venient. The  Kipp  hydrogen  generator  and  two  wash  bottles, 
one  containing  a  10  per  cent  solution  of  lead  nitrate,  the 
other  a  solution  of  silver  nitrate,  are  fastened  to  a  base  which 
is  placed  on  a  shelf  near  a  sink.  Some  form  of  a  Bunsen 
pump  is  placed  on  the  wall  above  the  sink.  Brass  tubing 
connects  the  water  pump  with  a  mercury  manometer  and 
with  the  gas  generator  by  means  of  a  Y-tube.  The  third  limb 
of  the  Y-tube  is  connected  to  the  vessel  in  which  the  cultures 
are  to  be  placed. 

In  the  bottom  of  a  wide-mouthed  bottle  is  placed  a  layer  of 
sand.  A  solution  of  pyrogallic  acid  is  poured  upon  the  sand. 
The  bottle  is  closed  by  a  rubber  stopper  provided  with  one 


> 


FIG.  15.  Ari'ARATrs  FOR  OBTAINING  ANAKROBIC  CONDITIONS 
(After  Wesbrook) 


83 


84 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


perforation  in  which  is  placed  a  glass  tube  carrying  a  ground- 
glass  stopcock.  The  stopcock  should  be  lubricated  with  vase- 
line or,  preferably,  a  saturated  solution  of  rubber  in  paraffin. 
The  rubber  stopper  should  be  coated  with  soft  paraffin  so 

r,  that  it  will  not  adhere  to  the  glass. 

/     k  Just  before  closing  the  bottle  a  small  amount 

of  a  10  per  cent  solution  of  potassium  hydrate 


is  added.  The  cock  in  the  tube  leading  to  the 
generator  is  closed,  and  the  air  is  exhausted 
from  the  vessel.  The  cock  between  the  pump 
and  the  Y-tube  is  then  closed,  while  the  one 
admitting  the  hydrogen  is  gradually  opened. 
When  the  jar  is  filled  with  hydrogen  the  ex- 
haustion is  repeated.  The  process  must  be 
repeated  five  or  six  times.  Any  slight  amount 
of  oxygen  remaining  will  be  absorbed  by  the 
pyrogallic  acid.  In  order  to  avoid  the  forcing 
of  the  stopper  from  the  jar,  due  to  the  expan- 
sion of  the  hydrogen  in  the  incubator,  a  nega- 
tive pressure  of  100—150  mm.  of  mercury 
should  be  obtained  by  partially  exhausting 
the  vessel. 

Other  more    simple    but  less    satisfactory 
methods   of    obtaining  anaerobic   conditions 

FIG  16    BURRI'S  w^   ^e  f°und   described    in   text-books    on 
CULTURE  TUBE     general  bacteriology. 

This  tube  allows      The  medium  employed  for  anaerobic  cul- 


without  the  use  of  Dextrose  is  usually  employed. 

In  order  to  avoid  the  trouble  connected 
with  the  use  of  Petri  dishes  in  anaerobic  work,  the  method 
devised  by  Burri  is  recommended  for  the  isolation  of  pure 


MILK  FERMENTATIONS  85 

cultures.  Instead  of  the  ordinary  culture  tube,  tubas  open  at 
both  ends  are  made  from  glass  tubing.  These  are  stoppered 
at  the  lower  end  with  rubber  stoppers,  but  otherwise  they  are 
treated  as  usual.  The  inoculation  of  the  medium  is  as  usual. 
Care  should  be  taken  to  heat  the  medium  for  some  time  in 
boiling  water  to  expel  the  dissolved  oxygen,  to  mix  very 
thoroughly  with  the  melted  medium  the  substance  to  be 
examined,  and  to  solidify  the  inoculated  medium  quickly. 

After  incubation,  those  tubes  which  do  not  show  too 
numerous  colonies,  and  from  which  it  is  desired  to  make 
subcultures,  have  the  agar  removed  by  withdrawing  the 
rubber  stopper  and  pushing  the  agar  core  into  a  sterile 
Petri  dish  by  means  of  a  sterile  glass  rod.  The  agar  can 
then  be  divided  with  a  sterile  knife,  so  as  to  render  any 
particular  colony  accessible. 

Exercise.  Each  student  will  heat  to  80°  C.  for  fifteen  minutes  a 
sample  of  milk  inoculated  with  a  small  amount  of  soil.  Incubate  at 
37°  C.  If  the  milk  undergoes  an  acid  fermentation,  isolate  the  causal 
organism  by  the  method  described.  Make  a  detailed  cultural,  morpho- 
logical, and  biochemical  study  pf  the  organism. 


CHAPTEE  V 

PRESERVATION  OF  MILK 

It  is  impossible  to  produce  milk  that  does  not  contain  a 
greater  or  less  number  of  bacteria,  since  some  of  the  sources 
of  contamination  are  of  such  a  nature  that  they  cannot  be 
wholly  avoided.  A  large  part  of  the  bacteria  that  gain  en- 
trance to  the  milk  find  it  a  favorable  nutrient  medium,  and 
products  are  produced  in  it  as  a  result  of  their  growth,  so 
that  it  is  rendered  more  or  less  unpalatable,  or  even  danger- 
ous as  human  food. 

The  period  during  which  milk  is  fit  for  use  is  usually  to 
be  measured  by  hours  rather  than  by  days.  When  the  milk 
is  consumed  at  or  near  the  place  of  production,  the  question 
of  preservation  is  of  no  great  economic  importance.  With 
the  growth  of  the  modern  city,  however,  the  zone  from  which 
the  milk  must  be  drawn  has  widened  until,  in  many  cases,  it 
is  transported  for  hundreds  of  miles  and  does  not  reach  the 
consumer  for  twenty-four  to  forty-eight  hours  after  with- 
drawal from  the  animal.  It  is  thus  exceedingly  desirable 
that,  so  far  as  possible,  bacterial  life  be  excluded  from  milk 
by  the  exercise  of  the  most  rigid  but  practicable  methods 
while  in  the  hands  of  the  producer.  It  is  also  quite  as 
necessary  that  the  milk  while  in  transit  be  kept  under  such 
conditions  as  will  check  bacterial  growth. 

While  the  partial  exclusion  of  bacteria  will  enhance  the 
keeping  quality  of  the  product,  milk  preservation  is  more  par- 
ticularly limited  to  those  methods  by  which  the  organisms 

86 


PRESERVATION  OF  MILK  87 

are  more  or  less  completely  inhibited  or  destroyed  after  they 
have  gained  access  to  the  milk. 

Various  chemical  and  physical  agents  are  made  use  of  to 
destroy  the  bacteria  in  milk  or  to  prevent  their  growth.  The 
chemicals  used  are  those  known  as  antiseptic  substances, — 
formalin,  borax,  boracic  acid,  or  proprietary  compounds  which 
contain  one  of  these  antiseptic  substances.  Other  substances, 
such  as  sodium  carbonate,  by  neutralizing  the  acid  produced, 
lengthen  the  time  before  the  milk  acquires  an  acid  taste. 
Others,  such  as  hydrogen  peroxide,  act  even  more  powerfully, 
destroying  the  bacteria  without  altering  the  taste  or  appear- 
ance of  the  milk,  if  they  are  not  used  in  too  large  amounts. 

The  use  of  practically  all  chemicals  for  the  preservation  of 
milk  is  classed  as  an  adulteration  and  is  forbidden  by  law. 
The  same  objection  cannot  be  urged  against  the  use  of  physi- 
cal agents,  such  as  heat  and  cold,  employed  separately  or  in 
combination.  The  attempt  has  been  made  to  freeze  the  milk 
wholly  or  in  part  (Casse  system),  but  this  process  has  not  met 
with  much  success.  Eefrigeration  or  icing,  so  as  to  lower  the 
temperature  to  4.5°  C.  or  below,  is  most  successful.  While  such 
temperatures  prevent  in  considerable  measure  the  growth  of 
the  lactic  organisms,  there  are  some  species  of  bacteria  capable 
of  multiplying  at  even  very  low  temperatures  (0-1.5°  C.).  In 
refrigerated  but  unfrozen  milk  these  may  develop  abundantly, 
although  usually  they  do  not  alter  the  normal  characteristics 
of  the  milk  (taste,  smell,  etc.)  to  any  great  extent. 

By  the  use  of  high  temperatures  it  is  possible  to  destroy 
effectively  the  bacteria  present.  The  temperature  at  which 
any  form  is  destroyed  is  known  as  its  thermal  death  point. 
This  temperature  varies  greatly  with  the  organism,  depend- 
ing upon  its  ability  to  produce  endospores.  The  exact  point 
is  also  correlated  with  the  length  of  time  for  which  the 


88  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

exposure  is  made.  Where  the  vegetative,  growing  organism  is 
subjected  to  heat,  it  is  killed  at  approximately  a  scalding 
temperature,  varying  from  60°  to  80°  C.,  depending  on  the 
period  of  exposure.  Endospores  cannot  be  destroyed  unless 
the  temperature  is  raised  to  the  boiling  point  or  above,  and 
the  exposure  continued  for  a  considerable  time.  No  process 
under  commercial  conditions  completely  destroys  all  traces 
of  germ  life,  so  that  subsequent  chilling  of  the  heated  milk 
is  necessary  in  order  to  prevent  rapid  growth  of  the  remain- 
ing cells. 

In  condensed  milk  the  main  factor  accounting  for  its  keep- 
ing qualities  is  the  concentrated  nature  of  the  liquid.  By  the 
application  of  heat  in  a  partial  vacuum  a  portion  of  the  water 
is  evaporated.  The  concentration  of  the  milk  is  further  in- 
creased by  the  addition  of  cane  sugar.  The  keeping  quality 
is  thus  dependent  upon  the  same  condition  that  is  found  in 
the  case  of  sirups,  which  "  work,"  i.e.  ferment,  unless  suffi- 
ciently concentrated.  By  heating  during  the  evaporation  many 
of  the  bacteria  are  killed.  Those  remaining  are  unable  to  mul- 
tiply until  the  milk  is  diluted  with  water. 

Exercise.  EFFECT  OF  CONCENTRATION  ON  BACTERIAL  GROWTH. 
Place  about  2  cc.  of  sweetened  condensed  milk  in  each  of  two  sterile 
test  tubes.  To  one  add  10  cc.  of  sterile  water.  Inoculate  both  tubes 
with  the  same  amount  of  a  culture  of  a  digesting  organism,  and  note 
changes  that  occur. 

Effect  of  temperature  on  bacterial  growth.  The  three 
temperatures  which  are  usually  employed  for  storage  of 
milk  under  practical  conditions  are  10°  C.  (the  temperature 
of  an  ordinary  ice  box),  20°  C.  (room  temperature),  and  30°- 
37°  C.  (temperatures  approximating  blood  heat,  as  when  a 
large  can  of  milk  fresh  from  the  cow  is  allowed  to  stand 
without  cooling). 


PRESERVATION  OF  MILK  89 

A  sample  of  perfectly  fresh  milk  should  be  poured  into 
three  sterile  flasks  and  incubated  at  10°  C.,  20°  C.,  and  37°  C. 
At  frequent  intervals  comparative  plate  cultures  should  be 
made  from  the  samples  by  the  loop  method,  in  order  to  de- 
termine the  relative  rapidity  of  growth  of  bacteria  at  the 
various  temperatures.  The  plates  should  be  made  at  least 
every  twenty-four  hours. 

Besides  this  approximate  quantitative  examination  a  de- 
termination of  the  rate  of  acid  development  should  be  made 
by  removing  5  cc.  of  each  sample  each  time  the  plates  are 
made,  and  determining  the  amount  of  acid  therein.  The  max- 
imum amount  of  acid  formed  at  the  various  temperatures 
should  be  determined,  and  the  nature  and  flavor  of  the  cur- 
dled milk  also  noted. 

Exercise.  Each  student  will  procure  a  sample  of  fresh  milk 
(100  cc.),  divide  the  same  between  three  flasks,  incubate  them  at 
the  temperatures  indicated,  and  at  intervals  of  twenty-four  hours 
prepare  lactose-agar  plates,  using  the  loop  method  of  dilution.  Con- 
tinue until  no  further  increase  in  acidity  is  noted. 

Pasteurization  of  milk.  A  large  proportion  of  the  bac- 
teria found  in  milk  are  vegetating  cells,  such  forms  making 
up  over  99  per  cent  of  the  bacterial  content  of  normal  milk. 
The  vegetating  cells  of  nearly  all  forms  of  bacteria  are  easily 
killed  at  temperatures  above  60°  C.  In  milk  thoroughly  pas- 
teurized only  the  spores  of  spore-forming  bacteria  are  left. 
To  attain  this  condition  it  is  necessary  to  heat  the  milk  at 
such  temperatures  and  for  such  periods  as  to  alter  its  chem- 
ical and  physical  nature  in  some  degree.  Some  of  the  changes 
are  as  follows :  The  fat  globules  in  normal  milk  are,  to  a  con- 
siderable extent,  in  clusters  or  masses,  which,  on  account  of 
having  a  greater  volume  for  a  given  surface,  are  able  to 
overcome  the  resistance  offered  by  the  milk  serum,  and  rise 


90  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

rapidly  to  the  surface.  If  the  clusters  are  broken  up  and  each 
globule  is  separated  from  every  other,  the  creaming  is  much 
less  rapid.  Heating  under  constant  agitation  breaks  up  the 
aggregates  of  fat  globules  and  injures  the  creaming  power  of 
the  milk.  If  the  individual  fat  globules  are  broken  into  minute 
fat  droplets,  as  in  the  case  of  the  so-called  "homogeneous 
milk,"  the  fat  cannot  be  removed,  even  by  passing  the  milk 
through  a  cream  separator.  The  viscosity  of  cream  is  dimin- 
ished when  heated  on  account  of  the  breaking  up  of  the 
aggregations  of  fat  globules. 

Milk  contains  a  substance  whose  nature  is  unknown, 
which  has  the  power  of  decomposing  hydrogen  peroxide. 
If  the  milk  is  heated  to  temperatures  above  80°  C.,  it  loses 
this  power.  This  property  is  made  use  of  in  the  Storch  test 
for  heated  milk. 

In  commercial  practice  two  methods  are  used  for  the 
pasteurization  of  milk.  They  do  not  differ  in  principle,  but 
simply  in  the  time  and  temperature  at  which  the  milk  is 
heated.  In  the  intermittent  or  discontinuous  method  the 
milk  is  heated  at  temperatures  from  60°  to  70°  C.  for  twenty 
or  thirty  minutes,  either  in  bottles  or  in  a  container,  so  ar- 
ranged that  the  milk  can  be  heated  rapidly  and  stirred  dur- 
ing the  process.  In  the  continuous  method  the  milk  is  heated 
almost  instantaneously  to  70°-90°  C.,  maintained  there  a  few 
seconds,  and  rapidly  cooled.  Many  types  of  apparatus  have 
been  devised  for  this  purpose,  none  of  which,  however,  are 
wholly  satisfactory. 

Exercise.  EXAMINATION  OF  MILK  PASTEURIZED  IN  A  DISCONTIN- 
UOUS MACHINE.  Each  student  will  procure  in  a  sterile  container  a 
sample  of  raw  milk  and  also  a  sample  of  the  same  milk  after  it  has 
been  pasteurized  by  the  intermittent  method  at  65°  C.,  and  subse- 
quently cooled.  Quantitative  lactose-agar  plates  should  be  made 


PRESERVATION  OF  MILK  91 

either  by  the  exact  dilution  method  or  by  the  loop  method.  Deter- 
mine the  percentage  of  bacteria  destroyed  by  the  pasteurizing  process. 

An  examination  should  also  be  made,  if  possible,  of  milk  before 
and  after  pasteurization  in  a  continuous  machine,  and  the  efficiency 
of  the  two  methods  compared. 

Exercise.  EFFECT  OF  HEAT  ON  THE  CREAMING  OF  MILK.  Heat 
some  whole  milk  over  a  water  bath  to  80°  C.  for  fifteen  'minutes, 
stirring  constantly  during  the  heating.  Cool  at  once  to  at  least  20°  C. 
by  placing  in  cold  water ;  compare  the  creaming  power  of  the  heated 
sample  with  that  of  raw  milk  by  placing  a  certain  amount  of  each  in 
large  test  tubes  or  graduated  cylinders  and  placing  in  the  ice  box  for 
twenty-four  hours. 

In  Denmark  and  in  several  of  the  states  of  the  United 
States  it  is  required  by  law  that  all  creamery  skim  milk  shall 
be  heated  to  80°  C.  before  it  can  be  returned  to  the  farms. 
This  regulation  is  for  the  purpose  of  preventing  the  spread  of 
contagious  diseases  such  as  tuberculosis  and  foot  and  mouth 
disease,  the  causal  organisms  of  which  are  often  found  in  the 
mixed  creamery  skim  milk.  In  order  to  determine  whether 
the  creameries  are  obeying  the  law,  some  means  of  control 
must  be  employed,  and  the  Storch  test  is  used  for  determin- 
ing whether  or  not  the  milk  has  been  heated  to  the  required 
temperature. 

Detection  of  heated  milk.  Storch 's  reaction.  The  test 
is  made  by  adding  to  5  cc.  of  milk  in  a  test  tube  one  drop 
of  a  0.3  per  cent  solution  of  hydrogen  peroxide  (the  commer- 
cial product  contains  3  per  cent  of  the  peroxide),  and  at  once 
adding  2  drops  of  a  2  per  cent  solution  of  paraphenylendia- 
min.  Mix  the  reagents  thoroughly  with  the  milk.  If  the 
milk  becomes  an  intense  blue  at  once,  either  it  has  not  been 
heated  at  all  or  if  heat  was  applied  it  was  not  higher  than 
78°  C.  If  the  color  appears  more  slowly,  after  the  lapse  of 
half  a  minute,  and  becomes  a  clear  grayish  blue,  the  milk 
has  been  heated  to  79°-80°  C.  If  the  milk  retains  its  white 


92  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

color  unchanged,  it  has  been  heated  above  80°  C.  By  the  use 
of  this  test  10  per  cent  of  milk  heated  to  78°  C.  can  be  de- 
tected when  mixed  with  milk  heated  above  80°  C. 

The  test  cannot  be  made  with  milk  in  which  the  acidity 
has  increased  to  any  extent.  In  the  testing  of  sour  milk  or 
buttermilk  the  sample  to  be  tested  must  first  be  neutralized. 
The  blue  color  does  not  develop  except  in  the  presence  of 
casein.  In  unheated  whey  a  brown  color  develops,  while  in 
whey  heated  above  80°  C.  no  color  is  produced  on  adding 
the  reagents. 

Exercise.  Heat  a  sample  of  milk  to  70°  C.  and  cool  at  once  to 
20°  C.  The  heating  should  be  done  in  such  a  manner  that  the  sample 
can  be  stirred  during  the  process,  so  as  to  insure  uniform  application 
of  heat  to  all  particles  of  the  milk.  Apply  Storch's  test  to  a  portion 
of  the  unheated  milk  and  to  the  heated  milk. 

Repeat  the  experiment  at  75°  and  80°  C.  Note  comparative  results. 


CHAPTER  VI 

RELATION  OF  BACTERIA  TO  BUTTER 

Effect  of  creaming  on  the  distribution  of  the  bacteria  in 
milk.  When  cream  is  separated  from  milk  in  a  centrifugal 
separator,  by  virtue  of  its  lighter  specific  gravity,  it  is  thrown 
toward  the  center  of  the  machine.  The  skim  milk  and  the 
heavier  insoluble  particles  which  constitute  the  sediment  that 
collects  on  the  wall  of  the  separator  bowl  are  of  course 
thrown  to  the  outside.  This  differentiation  of  the  milk  ele- 
ments also  exerts  a  material  influence  on  the  distribution  of 
bacterial  life  in  the  milk.  If  milk  were  wholly  liquid,  i.e. 
contained  none  of  its  normal  constituents  in  other  than  a 
soluble  condition,  the  distribution  of  the  bacteria  present  in 
the  milk  would  depend  entirely  upon  their  specific  gravity 
in  comparison  to  that  of  milk ;  but  as  there  are  insoluble  sub- 
stances present  which  are  materially  lighter  (fat),  as  well  as 
those  that  are  heavier  (casein  and  dirt),  than  the  milk  serum, 
the  effect  of  these  materials  modify  profoundly  the  distribu- 
tion of  the  bacteria.  Mud  and  silt  in  a  body  of  water  carry 
down  the  larger  part  of  the  microorganisms  that  may  be 
suspended  in  the  same.  So  in  a  similar  way  the  fat  globules 
and  the  separator  slime  will  be  found  to  act  as  differential 
agents  in  the  partial  separation  of  organisms.  The  immense 
number  of  fat  globules  passing  rapidly  toward  the  center  of 
the  separator  bowl  carry  with  them  mechanically  a  large  part 
of  the  bacteria  in  the  milk.  In  gravity  creaming  the  same 
phenomenon  occurs,  but  to  a  much  less  degree. 


94     EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Examination  of  cream  and  separator  slime.  The  quanti- 
tative and  qualitative  examination  of  cream  may  be  carried 
out  in  the  same  way  as  in  the  case  of  milk,  unless  the  cream 
is  very  thick,  in  which  case  the  measurement  by  means  of  a 
pipette  is  too  inaccurate.  A  definite  amount  of  cream  (10 
grams)  may  be  added  to  a  definite  amount  of  sterile  water 
in  a  flask  by  balancing  the  flask  on  the  scales  and  adding 
the  desired  amount  of  cream.  The  cream  should  then  be 
thoroughly  mixed  with  the  water  by  shaking. 

The  tenacity  of  separator  slime  is  such  that  it  can  be  handled 
only  as  a  solid.  A  definite  amount  is  weighed  out  by  placing 
on  the  pan  of  the  scales  a  piece  of  sterile  filter  paper  (9  cm. 
in  diameter),  and  upon  this  a  smaller  piece  (7  cm.  in  diame- 
ter). The  smaller  paper  with  the  slime  is  placed,  by  means 
of  sterile  forceps,  in  a  definite  amount  of  sterile  water  and 
shaken  until  the  slime  is  completely  disintegrated. 

Exercise.  Each  student  will  make  a  quantitative  examination  of 
(1)  skim  milk  ;  (2)  cream  ;  (3)  separator  slime. 

The  milk  should  not  have  an  acidity  of  more  than  0.2  per  cent. 
The  cream  separated  should  contain  at  least  30  and  preferably  40  per 
cent  of  fat.  The  plates  may  be  prepared  by  the  exact  dilution  method 
or  by  the  loop  method  (p.  28),  which  will  give  comparative  results. 

Examination  of  sweet  and  ripened  cream.  The  bacteria 
in  the  cream  find  favorable  conditions  for  growth  in  case  the 
cream  is  kept  at  ordinary  temperatures.  The  growth  is  con- 
fined largely  to  acid-producing  organisms  of  one  form  or 
another.  The  amount  of  acid  formed  will  depend  on  the 
richness  of  the  cream.  The  fat  is  to  be  looked  upon  as  an 
inert  body  so  far  as  acid  production  is  concerned.  In  deter- 
mining the  acid,  the  volume  taken  consists  of  fat  and  the 
liquid  part  of  the  cream.  In  the  latter  all  acid  is  developed ; 
hence  if  the  fat  makes  up  30  per  cent  of  the  total  volume, 


KKLA1ION  OF  BACTERIA  TO  BUTTER      95 

and  the  acidity  is  0.6  per  cent,  the  acidity  of  the  liquid  j>;irt 
of  the  cream  amounts  to  0.85  per  cent.  In  a  40  per  cent 
cream  it  will  be  found  impossible  to  develop  much  over  0.7 
per  cent  of  acid.  Such  an  increase  in  acid  can  only  occur 
when  the  bacteria  have  greatly  increased  in  number. 

Exercise.  Each  student  will  make  a  quantitative  and  qualitative 
examination  of  sweet  cream  and  ripened  cream.  The  sweet  cream 
should  be  that  obtained  from  very  fresh  milk.  The  ripened  cream 
should  have  developed  the  acidity  necessary  for  churning.  Prepare 
from  each,  by  the  loop  method,  lactose-litmus-agar  plates  and  also 
lactose-agar  plates.  Note  which  cream  shows  the  greatest  diversity 
of  species. 

Relation  of  butter  flavor  to  bacterial  development.  Two 
types  of  butter  are  made,  one  from  sweet  cream  and  the  other 
from  cream  which  has  been  allowed  to  develop  a  greater  or 
less  amount  of  acid,  —  ripened  cream.  These  types  of  butter 
differ  primarily  in  flavor.  Small  samples  of  cream  may  be 
readily  churned  in  Mason  fruit  jars.  The  jars  should  not  be 
filled  more  than  half  full,  and  may  be  shaken  by  hand  or 
in  a  shaking  machine.  As  nearly  as  possible  the  process 
should  be  the  same  as  that  employed  on  a  large  scale, 
washing,  salting,  etc. 

The  relation  of  flavor  to  bacterial  products  can  be  more 
specifically  shown  by  dividing  a  quantity  of  perfectly  sweet 
cream.  One  portion  is  to  be  clVurned  without  further  treat- 
ment ;  to  the  other  a  large  amount  of  starter  should  be  added 
(25-30  per  cent),  and  the  mixture  churned.  The  resulting 
butters  should  be  examined  as  to  flavor. 

Exercise.  The  samples  of  cream  examined  in  the  previous  exer- 
cise should  be  churned  and  the  flavor  of  the  butters  noted. 

Quantitative  analysis  of  butter.  Butter  is  much  more 
difficult  to  sample  than  milk  on  account  of  difficulty  in  se- 
curing a  thoroughly  even  emulsion.  Two  methods  are  used. 

^^ 

OF  THE     ' 

UNIVERSITY 


96  EXPERIMENTAL  DAIRY  BACTERIOLOGY 

1.  A  definite  quantity  of  the  butter  may  be  weighed,  as 
for  example,  1  gram.    If  the  butter  to  be  sampled  is  in  the 
form  of  a  pound  print,  a  piece  should  be  cut  from  one  end 
with  a  sterile  knife,  and  the  sample  taken  from  various  por- 
tions of  the  freshly  exposed  surface.    If  the  butter  is  in  a  tub 
or  jar,  the  sample  should  be  taken  with  a  sterile  trier,  the 
plug  thus  taken  split  with  a  knife,  and  portions  taken  from 
various  parts  of  the  plug.  The  butter  can  be  weighed  by  plac- 
ing on  the  pan  of  the  balance  a  circular  piece  (9  cm.)  of  sterile 
filter  paper.    Filter  papers  of  this  kind  should  be  sterilized  in 
a  Petri  dish  and  kept  in  stock.    On  the  larger  filter  place  a 
7  cm.  filter  paper,  on  which  the  butter  is  to  be  placed.    The 
contamination  from  the  air  during  the  weighing  process  will 
be  of  no  appreciable  importance.    The  smaller  filter  with 
the  butter  should  be  transferred  by  means  of  sterile  forceps 
to  a  definite  volume  of  sterile  water,  which  is  then  heated 
from  40°  to  45°  C.  in  order  to  melt  the  butter  and  to  distrib- 
ute the  bacteria  uniformly.   The  sample  for  further  dilution 
should  be  removed  while  the  contents  of  the  flask  are  well 
emulsified. 

2.  Instead  of  weighing  the  butter  it  may  be  melted  at 
40°-45°  C.  and  1  cc.  removed  with  a  warm  pipette  and  trans- 
ferred to  warm  sterile  water.  The  pipette  should  be  freed  from 
the  adherent  fat  by  filling  it  with  the  dilution  water  a  number 
of  times.    Further  steps  in  the  preparation  of  the  plate  cul- 
tures and  in  their  study  are  carried  out  as  described  in  the 
case  of  the  examination  of  milk. 

Relation  of  age  of  butter  to  bacterial  content.  Butter  is 
made  up  mainly  of  butter  fat,  water  saturated  with  salt,  and 
small  quantities  of  the  other  ingredients  of  milk.  It  contains 
but  little  proteid  matter,  and  is  therefore  not  well  suited  for 
bacterial  growth,  The  difference  between  the  germ  content 


RELATION  OF  BACTERIA  TO  BUTTER      97 

of  the  fresh  butter  aud  the  same  butter  some  weeks  later 
should  be  determined. 

Exercise.  Each  student  will  examine  quantitatively  a  sample  of 
fresh  creamery  butter.  The  portion  sampled  is  to  be  placed  in  the 
refrigerator  and  reexamined  three  or  four  weeks  later. 

Starters.  With  increasing  knowledge  of  bacteria  and  of 
the  various  kinds  finding  favorable  conditions  for  growth  in 
milk  and  cream,  it  became  evident  that  the  flavor  of  the 
butter  depended  largely  upon  the  types  of  bacteria  present. 
Tn  order  to  emphasize  the  effect  of  specific  forms  on  the  flavor 
of  the  product,  it  is  necessary  to  insure  their  predominance  in 
the  cream  by  the  addition  of  pure  cultures.  The  three  types 
most  commonly  present  in  cream  are:  (1)  lactic-acid-forming 
organisms  of  the  desirable  type  (B.  lactis  acidi  Leichmann) ; 
(2)  acid-producing  organisms  of  less  desirable  types  (B.  coli 
communis,  B.  lactis  aerogenes) ;  (3)  organisms  liquefying  casein 
(B.  fluorescens  liquefaciens,  etc.). 

Pasteurized  or  perfectly  sweet  fresh  cream  should  be  in- 
oculated with  pure  cultures  of  the  above  types  of  bacteria, 
both  alone  and  in  mixtures.  The  growth  should  be  allowed 
to  continue  for  some  time  and  the  flavor  of  the  ripened  cream 
noted.  The  cream  should  be  churned  and  the  flavor  of  the 
butter  determined. 

These  trials  may  be  carried  out  under  more  practical  con- 
ditions by  preparing  the  various  starters  in  the  laboratory, 
and  the  remaining  portion  of  the  work  should  then  be  done 
under  creamery  conditions. 

Commercial  starters.  With  the  determination  of  the  im- 
portance of  the  types  of  bacteria  in  influencing  the  flavor  of 
the  butter,  it  became  evident  that  selected  forms  should  be 
grown  in  masses  and  added  to  the  cream  to  insure  their 
predominance.  The  introduction  of  the  "  pure  culture  "  into 


98  EXPERIMENTAL   DAIRY  BACTERIOLOGY 

butter  making  was  largely  due  to  Storch  and  to  Weigmann. 
Beginning  in  1890,  the  use  of  pure  cultures  has  spread  rapidly 
and  is  now  quite  universally  employed  in  commercial  butter 
production. 

On  account  of  the  difficulty  and  trouble  the  butter  maker 
encounters  in  selecting  milk  for  the  preparation  of  home- 
made starters,  and  the  lack  of  uniformity  of  product  con- 
nected with  their  use,  the  majority  of  butter  makers  prefer 
to  purchase  the  starter  foundation.  A  number  of  firms  are 
engaged  in  the  preparation  and  sale  of  pure  culture  or  com- 
mercial starters  for  butter  making. 

These  cultures  are  sold  in  liquid  and  dry  forms.  The 
liquid  starters  usually  consist  of  a  milk  culture  of  the  organ- 
ism. The  dry  starters  are  prepared  by  mixing  with  a  liquid 
culture  in  an  actively  growing  condition  some  inert  sub- 
stance, as  milk  sugar,  milk  powder,  etc.,  in  order  to  absorb 
the  moisture  of  the  culture  and  thus  maintain  the  organ- 
isms in  an  active  condition.  In  liquid  cultures  deterioration 
is  rapid  on  account  of  the  injurious  effect  of  the  by-products 
of  the  bacteria  themselves.  In  dry  cultures  deterioration  is 
due  mainly  to  the  influence  of  desiccation  on  the  contained 
organisms.  The  effect  of  desiccation  on  most  lactic  bacteria 
is  much  less  than  that  of  the  by-products  (acid) ;  hence 
the  starters  in  dry  form  can  be  kept  much  longer  than  in 
liquid  form. 

The  important  points  to  be  noted  in  the  examination  of 
commercial  starters  are :  (1)  purity ;  (2)  vitality  and  fermen- 
tative power ;  (3)  preservation  of  original  properties  on  prop- 
agation ;  (4)  flavor-producing  properties. 

Purity.  The  purity  of  a  starter  may  be  tested  by  preparing 
from  the  original  package  lactose-agar  and  plain  agar  plate 
cultures.  A  series  of  dilutions  should  be  made  in  order  to 


KKLATION  OF  BACTERIA   TO  BUTTER  99 

insure  plates  with  a  desirable  number  of  colonies,  so  that  all 
forms  present  shall  have  an  opportunity  to  develop. 

From  liquid  starters  plates  are  prepared  as  in  testing  the 
purity  of  a  culture  (p.  31).  A  portion  of  the  dry  starter  may 
be  transferred  to  sterile  water  by  dipping  the  platinum  loop 
into  the  water  and  then  into  the  powder,  in  which  case  the 
moisture  will  cause  a  considerable  amount  of  the  powder  to 
adhere.  From  the  water  blank  which  has  been  well  shaken 
after  addition  of  the  powder,  plates  are  made  in  the  usual 
way. 

The  plates  should  be  carefully  studied,  the  different  types 
of  organisms  isolated,  and  a  detailed  study  made  of  each. 

It  should  be  remembered  that  a  dry  commercial  starter  is 
not  necessarily  to  be  condemned  because  it  is  not  absolutely 
pure.  When  propagated  the  lactic  organism  will,  if  properly 
chosen,  develop  so  much  more  rapidly  than  the  contaminat- 
ing organisms  that  these  will  not  exert  any  continued  effect 
in  the  starter  or  in  the  cream. 

Vitality  and  fermentative  power.  In  order  to  be  desirable 
for  starter  purposes,  an  organism  must  grow  rapidly  when 
inoculated  into  milk,  which  is  then  kept  at  temperatures 
ranging  from  20°  to  30°  C.;  it  must  also  produce  acid  rapidly, 
and  in  sufficient  amounts  for  the  production  of  the  desired 
degree  of  flavor,  and  to  insure  exhaustive  churning. 

Since  the  organisms  in  the  starter  as  purchased  may  be 
few  in  number  and  weakened  by  desiccation  or  by  the  ac- 
cumulation of  acid,  these  points  should  not  be  determined 
by  means  of  cultures  inoculated  directly  from  the  original 
package,  but  the  organisms  should  be  brought  to  a  normal 
state  of  vitality  by  inoculating  sterile  milk  from  the  original 
starter  and  incubating  for  twenty-four  hours.  From  this  pre- 
pare a  second  culture,  and  from  this,  after  twenty-four  hours' 


100         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

incubation,  may  be  inoculated  cultures  in  order  to  determine 
the  ability  of  the  organism  to  produce  acid.  Into  a  series  of 
flasks  containing  a  definite  amount  of  sterile  milk  are  inocu- 
lated varying  amounts  of  the  fresh  twenty-four-hour-old  milk 
culture  (1/100  cc.,  1/1000  cc.,  1/10,000  cc.  for  100  cc.  of 
milk).  The  flasks  should  be  kept  at  25°-30°  C.  With  a  cul- 
ture of  high  fermentative  power  1  part  to  1000  of  milk 
should  produce  curdling  in  twenty -four  hours  at  30°  C. 

The  amount  of  acid  in  the  curdled  milk  should  be  de- 
termined by  titration  with  N/20  sodium  hydroxide. 

Preservation  of  properties  on  propagation.  Many  lactic- 
acid  cultures  when  first  isolated  from  milk  show  desirable 
properties  for  use  in  a  starter,  but  on  continued  propagation 
lose  them  more  or  less  rapidly.  They  may  cease  to  produce 
sufficient  acid,  or  they  may  form  it  very  slowly.  Manifestly, 
such  organisms  are  not  well  adapted  for  starter  propagation. 
Their  flavor-producing  properties  may  deteriorate,  or  they  may 
commence  to  produce  a  slimy  or  ropy  fermentation  in  the 
milk.  The  ability  of  an  organism  to  preserve  its  original 
properties  can  only  be  determined  by  continued  propagation 
under  practical  conditions,  that  is,  in  the  same  manner  as 
would  be  done  in  a  creamery  in  the  carrying  on  of  mother 
starters  in  small  quantities.  The  propagation  should  be  carried 
out  under  definite  conditions  as  to  temperature  of  incubation 
and  the  degree  of  acidity  developed  in  the  starters. 

Flavor-producing  properties.  The  ability  of  the  organism 
to  produce  a  desirable  flavor  in  the  starter  and  in  the  butter 
may  be  determined  by  frequent  examinations  of  the  starters 
as  propagated.  The  students  should  be  drilled  in  the  judging 
of  starters. 

Exercise.  Each  student  will  make  a  detailed  study  of  a  commer- 
cial starter,  as  to  its  purity,  vitality,  flavor-producing  properties,  etc. 


RELATION  OF  BACTERIA  TO  BUTTER     101 

Relation  of  bacteria  to  keeping  quality  of  butter.  In 
butter  from  ripened  cream  the  bacterial  content  is  made  up 
almost  entirely  of  lactic-acid  bacteria.  During  the  ripening 
of  the  cream  the  other  types  of  bacteria  are  overgrown  by 
the  lactic-acid  type,  or  are  even  destroyed  by  the  acid  pro- 
duced. In  pasteurized-cream  butter  the  percentage  of  lactic 
bacteria  reaches  its  maximum,  and  the  pasteurized  product 
has  the  best  keeping  qualities.  In  butter  from  sweet  cream 
the  bacterial  content  consists  of  many  forms,  the  lactic  bac- 
teria being  few  in  number,  and  the  keeping  quality  of  the 
butter  is  impaired. 

Exercise.  The  samples  of  butter  prepared  in  the  exercise  on 
page  95  should  be  stored  in  closed  vessels  in  an  ordinary  refriger- 
ator and  examined  at  intervals  of  ten  days,  in  order  to  determine 
the  rate  of  deterioration  of  the  sweet-cream  butter  in  comparison 
with  that  of  butter  from  the  ripened  cream. 

Relation  of  bacteria  in  wash  water  to  butter.  During 
the  washing  of  the  butter  the  buttermilk  is  largely  replaced 
by  water  with  its  own  peculiar  bacterial  flora.  The  germ  con- 
tent of  wash  waters,  qualitatively  and  quantitatively,  varies 
greatly,  depending  upon  the  source,  manner  of  storage,  etc. 
Water  from  a  deep  well  protected  from  all  surface  drainage,  or 
from  a  protected  spring,  contains  the  minimum  of  bacteria, 
and  will  exert  bub  little  influence  on  the  quality  of  the  butter. 

Water  from  streams,  shallow  wells,  or  any  source  unpro- 
tected from  surface  water  contains  many  bacteria  and  usually 
of  kinds  injurious  to  the  butter.  On  storage  in  tanks  and 
reservoirs  the  bacterial  content  of  deep-well  and  spring  waters 
rapidly  increases.  This  is  especially  true  -when  the  storage 
tank  is  not  kept  perfectly  clean.  Water  freshly  pumped  is 
more  desirable  for  use  in  the  dairy  than  after  storage  for 
any  length  of  time. 


102          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

It  is  probable  that  water  which  would  be  pronounced  good 
as  the  result  of  a  sanitary  analysis  would  be  desirable  for  use 
in  the  dairy..  For  the  methods  of  making  a  complete  bacterio- 
logical analysis  of  water,  the  student  is  referred  to  books  on 
water  analysis. 

The  quality  of  the  water  may  be  determined  roughly  by 
the  inoculation  of  dextrose-fermentation  tubes  with  0.1,  1, 
and  10  cc.  of  the  water.  Gas  production  is  indicative  of  pol- 
lution with  organic  matter,  a  condition  which  should  not  ob- 
tain in  a  creamery  well.  The  results  obtained  with  the  series 
of  fermentation  tubes  give  some  idea  of  the  number  of  gas- 
producing  bacteria  present. 

Gelatin  plates  inoculated  with  varying  quantities  of  the 
water  (0.1,  0.5,  and  1  cc.)  should  show  few  liquefying  bac- 
teria, especially  of  the  fluorescent  type  (B.  fluorescens  lique- 
faciens). 

Flasks  of  sterile  milk  (100  cc.)  should  be  inoculated  with 
10  cc.  of  the  water,  and  the  nature  of  the  curd  and  the  flavor 
noted. 

Exercise.  Each  student  will  test  by  means  of  dextrose-fermenta- 
tion tubes,  gelatin  plates,  and  milk,  five  samples  of  water  representing 
supposedly  good  waters  and  also  those  polluted  with  varying  amounts 
of  surface  drainage. 


CHAPTER  VII 

RELATION  OF  BACTERIA  TO  CHEESE 

In  the  making  and  curing  of  cheese  of  all  kinds,  micro- 
organisms, and  especially  the  bacteria,  function  as  essential 
factors.  Both  cheese  made  with  rennet,  as  well  as  that  pro- 
duced from  curd  precipitated  by  acid,  are  markedly  affected 
by  the  activity  of  these  organisms. 

Ripening  of  cheese.  Cheese  differs  from  butter  in  that  it 
must  undergo  a  profound  series  of  changes,  physical  and 
chemical  in  nature,  before  it  is  fit  for  consumption.  These 
changes,  collectively  known  as  the  curing  or  the  ripening, 
are  exceedingly  complex,  and  differ  in  details  in  various  tyj  efl 
of  cheese,  but  possess  in  common  certain  characteristics,  the 
most  important  of  which  are  (1)  the  gradual  change  of  the 
insoluble  paracasein  to  a  series  of  compounds  more  or  less 
soluble  in  water,  and  hence  more  readily  digestible  than  the 
original  paracasein ;  (2)  the  production  of  substances  having 
more  or  less  distinctive  flavors  and  odors. 

Our  knowledge  relating  to  the  exact  nature  of  these  proc- 
esses is  yet  far  from  complete.  Moreover,  they  are  of  such  a 
complex  nature  that  they  cannot  be  readily  demonstrated 
by  laboratory  exercises.  Consequently  only  the  general  prin- 
ciples here  involved  can  be  outlined. 

The  gradual  change  of  the  insoluble  paracasein  to  soluble 
compounds  is  a  process  related  to  digestion,  and  undoubtedly 
produced  by  proteolytic  enzymes  from  various  sources.  Ren- 
net is  added  to  milk  primarily  for  the  purpose  of  curdling 

103 


104          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

it.  This  effect  is  due  to  the  presence  in  the  rennet  extract 
of  the  enzyme  rennin,  or  caseinase,  according  to  modern  termi- 
nology. Rennet  extract  also  contains  pepsin  (acid  proteinase), 
which  exerts  its  peculiar  action  when  acid  is  present,  as  is 
normally  the  case  in  cheese,  due  to  the  action  of  the  lactic- 
acid  bacteria  on  the  milk  sugar  of  the  whey.  An  increase 
in  the  amount  of  rennet  used  in  cheese  making  has  been 
found  to  hasten  the  rate  at  which  the  water-soluble  products 
are  formed,  and  hence  the  rapidity  of  ripening. 

In  addition  to  pepsin  there  is  an  enzyme  normal  to  milk, 
known  as  galactase  (alkali  proteinase),  which  also  exerts  a 
proteolytic  action  on  casein  or  paracasein.  What  r61e  the 
bacteria  play  in  the  gradually  increasing  solubility  of  the 
curd  is  not  definitely  determined. 

Role  of  acid-forming  bacteria.  In  cheddar  cheese  the 
initial  stages  of  ripening  are  closely  related  to  the  develop- 
ment of  the  lactic-acid  group  of  bacteria,  as  indicated  in 
the  following  experiment. 

Exercise.  Melt  two  tubes  of  lactose  agar,  cool  to  50°  C.,  and  add 
to  each  tube  about  15  per  cent  of  sterile  skim  milk,  which  is  well 
mixed  with  the  melted  agar.  If  the  milk  is  added  to  the  hot  agar, 
the  casein  of  the  milk  will  be  precipitated  in  flocks.  Pour  one  tube 
of  the  mixed  agar  and  milk  into  a  sterile  Petri  dish  and  allow  it  to 
solidify.  Inoculate  the  remaining  tube  heavily  from  a  pure  culture 
of  a  lactic-acid  organism  and  pour  into  a  Petri  dish.  Keep  both 
plates  for  twenty -four  hours  at  temperatures  favorable  for  the  growth 
of  the  lactic-acid  organism  used.  At  the  end  of  the  period  of  incu- 
bation place  on  the  surface  of  each  plate  a  strip  of  filter  paper  mois- 
tened with  rennet  extract.  Incubate  the  plates  at  37°  C.  for  from  two 
to  four  hours.  At  the  expiration  of  this  period  the  following  changes 
are  to  be  noted  :  On  the  uninoculated  plate  the  opacity  produced  by 
the  milk  is  increased  in  the  immediate  neighborhood  of  the  paper, 
due  to  a  physical  change  in  the  casein.  On  the  inoculated  plate  the 
casein  is  changed  to  soluble  compounds,  and  the  opacity  is  thus  de- 
stroyed in  the  immediate  neighborhood  of  the  paper.  Between  the 


RELATION  OF  BACTERIA   TO  CHEESE  105 

clear  zone  and  the  unchanged  part  of  the  medium  will  be  noted  a 
zone  of  increased  opacity  similar  to  that  produced  on  the  uninocu- 
lated  plate. 

Analysis  of  cheese.  As  was  pointed  out  in  the  discussion 
on  general  quantitative  methods,  it  is  impossible  to  determine 
in  any  sample  the  absolute  number  of  bacteria  present.  In 
the  curd  and  in  the  cheese  the  bacteria  are  held  in  place 
exactly  as  in  a  gelatin  or  agar  plate.  If  growth  occurs,  colo- 
nies will  result.  Thus  the  distribution  of  bacteria  in  cheese 
is  not  uniform,  but  in  masses.  In  the  preparation  of  plate 
cultures  it  is  essential  that  as  far  as  possible  the  masses  of 
bacteria  shall  be  disintegrated.  This  is  done  by  grinding  the 
cheese  with  some  sharp  crystalline  substance,  as  sand,  finely 
ground  glass,  or  sugar.  The  mixture  of  cheese  and  grinding 
material  is  suspended  in  water  and  well  shaken,  and  plates 
made  therefrom. 

Sampling.  If  an  uncut  cheese  is  to  be  sampled,  a  plug 
must  be  taken  with  a  cheese  trier.  The  rind  over  the  part 
to  be  sampled  should  be  removed  with  a  knife,  and  a  plug 
taken  with  a  sterile  trier  and  transferred  to  a  large  test  tube 
or  -flask.  The  trier  may  be  sterilized  by  heating  in  the  flame 
of  a  gas  burner  or  of  an  alcohol  lamp,  or  by  dipping  in  alcohol 
and  igniting  it.  If  a  cut  cheese  is  to  be  sampled,  an  interior 
piece  should  be  removed,  under  aseptic  conditions,  with  a  knife 
and  placed  in  a  sterile  container.  The  plug  .or  piece  of  cheese 
is  split  with  a  sterile  instrument,  and  pieces  removed  at  vari- 
ous places.  A  piece  of  sterile  filter  paper  is  placed  on  the 
pan  of  a  balance ;  on  this  piece  of  paper  is  placed  a  smaller 
one,  also  sterile,  on  which  may  be  placed  the  cheese  for 
weighing.  Exactly  1  gram  is  weighed  out  and  transferred 
to  a  small  porcelain  mortar,  10  grams  of  fine  sterile  quartz 
sand  are  added,  and  the  mixture  is  ground  until  the  cheese 


106          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

is  disintegrated  as  completely  as  possible,  when  it  is  trans- 
ferred to  a  flask  of  water  by  means  of  a  sterile  spatula,  or 
rubber-tipped  rod. 

The  flask  is  well  shaken,  to  free  the  sand  from  cheese  as 
far  as  possible.  In  transferring  with  a  pipette  a  portion  of 
this  suspension  to  other  water  blanks  the  sample  should  be 
taken  immediately  after  shaking,  before  the  sand  has  set- 
tled. Settling  may  be  prevented  by  holding  the  pipette  in  a 
horizontal  position  until  ready  to  deliver  the  contents.  The 
grinding  material  should  be  fine  enough  to  avoid  clogging 
the  pipette.  All  the  instruments  and  material  with  which 
the  cheese  comes  in  contact  should  be  sterile ;  the  contami- 
nation from  the  air  during  the  grinding  is  so  small  as  to 
be  negligible. 

From  the  various  dilutions  lactose-agar  plates  are  prepared. 
The  maximum  numbers  of  bacteria  are  found  in  cheese  from 
twenty-four  to  forty-eight  hours  after  making.  The  number 
decreases  rapidly  for  some  days,  reaching  at  last  a  level  below 
which  farther  decrease  is  very  slow.  In  cheese  twenty-four 
hours  old  over  a  billion  bacteria  per  gram  may  be  found. 

Exercise.  Each  student  will  examine  quantitatively  samples  of 
cheese  from  one  to  two,  from  ten  to  fifteen,  and  from  sixty  to  one 
hundred  days  old. 

Qualitative  examination  of  milk  for  cheese  making.  In 
milk  imended  for  cheese  manufacture  it  is  desirable  that  acid- 
forming  organisms  of  the  B.  lactis  acidi  type  shall  be  the  pre- 
dominating organisms.  As  is  known,  this  organism  produces 
no  gas  in  the  fermentation  of  lactose,  the  curd  produced  be- 
ing perfectly  homogeneous  and  pleasant  in  flavor  and  odor. 
Organisms  of  the  B.  coli  communis  and  B.  lactis  aerogenes 
types  are  detrimental  in  cheese,  since  they  cause  the  cheese 
to  be  filled  with  a  greater  or  less  number  of  holes  produced 


RELATION  OF  BACTERIA  TO  CHEESfi  107 

by  the  gas  formed  from  the  lactose.  The  flavor  of  the  cheese 
is  also  injured  because  of  other  by-products  of  the  acid 
fermentation. 

Tests  for  the  quality  of  the  milk  to  be  used  in  the  manu- 
facture of  cheese  have  for  one  of  their  purposes  the  detection 
of  the  second  class  of  organisms,  the  gas-forming  bacteria.  If 
a  sample  of  the  milk  is  placed  in  a  container  and  incubated 
at  37°  C.  for  a  few  hours,  until  curdling  has  taken  place,  the 
quality  of  the  milk  will  be  revealed  by  the  nature  of  the  curd. 
This  forms  the  so-called  Gerber  fermentation  test,  largely  used 
in  Germany  and  Switzerland. 

A  more  delicate  method  is  known  as  the  Wisconsin  Curd 
Test,  and  is  carried  out  as  follows :  Pint  jars  are  filled  with 
the  milk  to  be  tested.  The  jars  should  be  sterilized  before 
being  filled,  and  care  should  be  taken  not  to  contaminate  one 
sample  from  others,  as  by  the  use  of  dippers,  etc.  The  jars  of 
milk  are  warmed  to  37°  C.,  0.5  cc.  of  rennet  extract  is  added  to 
each  jar,  and  the  whole  is  well  mixed.  The  curd  is  cut  with 
a  case  knife.  The  jars  are  kept  at  37°-40°C.,  in  order  to 
facilitate  the  shrinking  of  the  curd,  as  well  as  the  growth  of 
the  gas-generating  bacteria.  After  the  curd  is  firm  the  whey 
is  poured  off.  Additional  portions  of  whey  will  be  expressed 
and  should  also  be  removed.  The  jars  are  kept  at  the  same 
temperature  for  eight  to  twelve  hours,  and  are  then  examined 
for  flavor,  texture,  and  presence  of  gas.  The  particulate  bodies 
in  the  milk,  fat  globules  and  bacteria,  are  retained  in  the  curd. 
As  the  curd  shrinks  until  it  occupies  but  about  one  sixth  of 
the  volume  of  the  milk,  the  bacteria  are  concentrated,  and 
the  changes  produced  by  them  are  more  apparent.  A  few  gas 
holes  scattered  through  the  total  volume  of  milk,  such  as  is 
occupied  by  the  curd  when  produced  by  the  acid-forming  bac- 
teria, might  pass  unnoticed,  but,  if  present  in  a  small  mass  of 


108          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

curd,  would  be  more  evident.  The  curd  mass  in  the  Wiscon- 
sin Curd  Test  can  also  be  removed  from  the  jar,  its  texture 
determined,  the  nature  of  the  surface  of  the  curd  noted,  and 
thus  the  presence  of  undesirable  bacteria  detected. 

This  test  for  the  quality  of  milk  is  also  used  in  the  exami- 
nation of  market  milks.  It  should  be  remembered  that  a  milk 
of  very  high  grade  may  give  poor  results  when  tested  in  this 
manner,  especially  if  the  results  are  interpreted  from  the 
usual  standpoint.  A  curd  of  fine  texture  is  only  possible 
when  the  milk  contains  a  considerable  number  of  lactic 
organisms,  since  the  acid  they  produce  changes  the  curd  so 
that  the  small  pieces  gradually  fuse  into  one  mass  and  show 
the  flaky  texture  desired  by  the  cheese  maker.  With  a  milk 
produced  under  such  conditions  as  to  exclude  the  lactic  bac- 
teria (certified  and  sanitary  milk),  the  matting  of  curd  will 
be  largely  prevented  and  the  texture  will  be  faulty.  On  the 
other  hand,  a  milk  of  poor  quality  from  the  standpoint  of 
the  milk  consumer,  on  account  of  its  high  bacterial  content 
and  consequently  poor  keeping  qualities,  may  produce  a  firm, 
solid  curd,  free  from  gas  holes. 

Each  student  should  make  curd  tests  from  various  samples 
of  milk.  In  case  milks  illustrating  the  various  types  cannot 
be  obtained,  they  may  be  prepared  by  inoculating  fresh  milk 
with  pure  cultures  of  lactic-acid  organisms ;  gas-forming  and 
liquefying  bacterial  mixtures  may  also  be  used.  Gas-form- 
ing types  are  readily  secured  by  adding  a  small  quantity  .of 
manure  to  milk.  The  milk  should  be  inoculated  and  allowed 
to  stand  over  night,  in  order  to  give  a  chance  for  the  inocu- 
lated organisms  to  develop  before  the  curd  tests  are  made. 
If  facilities  permit,  the  instructor  can  have  a  small  cheese  (10 
pounds  or  so)  made  from  milk  contaminated  with  gas-form- 
ing organisms,  so  that  the  course  of  changes  in  the  cheese 


RELATION  OF  BACTERIA   TO  CHEESE  109 

itself  can  be  followed.  In  such  cases  it  is  advisable  to  infect 
the  milk  on  the  farm  rather  than  at  the  factory.  This  can 
readily  be  done  by  placing  in  milk  cans  a  small  quantity 
of  a  gassy  starter,  so  that  the  normal  souring  organisms  are 
overpowered. 

Exercise.   Each  student  will  make  curd  tests  of  the  samples  of 
milk  furnished  him. 


CHAPTER   VIII 

MILK  HYGIENE 

Milk  as  a  distributer  of  disease.  Milk  frequently  serves 
as  an  agent  for  the  transmission  of  communicable  diseases. 
Such  distribution  may  be  from  one  animal  to  another  of  the 
same  or  different  species ;  from  animal  to  man,  when  the 
disease  is  one  common  to  both ;  from  man  to  man,  in  case  of 
diseases  that  do  not  affect  the  cow,  but  whose  nature  is  such 
that  milk  may  be  infected  with  the  causal  organisms. 

The  most  important  of  the  diseases  common  to  man  and 
cattle  is  tuberculosis.  The  milk  may  contain  the  tubercle 
bacilli  before  being  drawn  from  the  udder,  as  when  the  mam- 
mary gland  is  one  of  the  organs  affected,  and  even  when  no 
disease  can  be  detected  in  the  udder.  The  milk  may  be  in- 
fected after  its  withdrawal,  through  the  pollution  with  dust 
and  manure  during  the  milking  process. 

In  tuberculosis  of  the  lungs  the  tubercular  abscesses  not 
infrequently  break  and  discharge  their  purulent  contents  into 
the  air  passages.  This  material  is  coughed  up  and  swallowed 
by  the  animal.  During  the  coughing  a  small  part  is  ejected 
from  the  mouth.  In  the  passage  through  the  alimentary  tract 
the  tubercle  bacilli  are  uninjured  by  the  digestive  juices,  the 
material  of  which  the  sputum  is  composed  is  digested,  and 
the  tubercle  bacilli  are  freed  and  mixed  intimately  with  the 
feces.  During  the  milking  process  more  or  less  manure 
gains  entrance  to  the  milk,  and  may  infect  it  with  the 
disease-producing  organisms. 

110 


MILK  HYGIENE  111 

But  one  other  disease  common  to  man  and  cattle  is  of 
marked  importance  because  of  its  direct  transmission  by 
means  of  milk.  Foot  and  mouth  disease,  the  causal  organism 
of  which  is  unknown,  is  transmitted  by  milk.  The  milk  is 
infected  before  it  is  drawn. 

A  considerable  number  of  epidemics  of  typhoid  fever  and 
diphtheria  have  been  traced  to  infected  milk  supplies.  The 
organisms  causing  these  diseases  always  gain  entrance  to  the 
milk  after  its  withdrawal  from  the  animal.  The  farm  water 
may  become  contaminated  with  typhoid  organisms,  and  the 
use  of  such  water  for  washing  and  rinsing  of  dairy  utensils 
may  serve  to  contaminate  the  milk  supply. 

Convalescents  from  an  attack  of  the  fever  may  be  sources 
of  contamination,  if  they  are  concerned  in  the  handling  of 
the  milk,  or  persons  serving  in  the  dual  capacity  of  nurse 
and  milkman  may  be  the  means  of  infection.  An  attack  of 
typhoid  fever  may  be  so  slight  as  to  pass  unnoticed.  Such 
cases  known  as  w  walking  typhoid  "  are  especially  dangerous, 
since  the  individual  does  not  recognize  the  gravity  of  the 
situation  and  accordingly  does  not  use  precautionary  meas- 
ures to  prevent  infection  of  the  milk  supply. 

Another  factor  is  the  "  bacillus  carrier,"  a  person  who  may 
have  had  typhoid  fever  years  before,  but  who  still  harbors  in 
and  distributes  from  his  body  myriads  of  typhoid  bacteria. 
Such  persons  are  especially  dangerous,  as  they  themselves  do 
not  realize  the  trouble  they  may  cause.  The  presence  of  the 
organism  can  only  be  determined  by  a  thorough  examination 
of  the  dejecta  of  the  individual.  It  is  known  that  in  one  case 
they  persisted  in  the  body  for  over  forty-two  years. 

Diphtheria  bacilli  in  milk  may  be  traced  to  convalescing 
patients,  or  to  persons  having  to  do  with  diphtheria  patients 
and  the  milk  supply. 


112         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

It  is  useless  to  examine  milk  for  the  presence  of  the  organ- 
isms of  diphtheria  or  typhoid  fever.  They  do  not  find  favor- 
able conditions  for  luxuriant  growth  in  milk,  in  competition 
with  the  ordinary  milk  forms.  Again,  by  the  time  the  milk 
supply  is  suspected  of  being  the  agent  of  transmission,  the 
source  of  contamination  has  often  disappeared  and  the  milk 
is  no  longer  dangerous.  In  order  to  determine  the  source 
of  an  epidemic,  a  careful  historical  survey  of  all  the  condi- 
tions surrounding  the  outbreak  must  be  made,  rather  than 
to  seek  the  causal  organism  in  the  water  or  milk  supply  by 
bacteriological  methods. 

Tubercle  bacilli  cannot  grow  in  milk  after  withdrawal  from 
the  animal,  but  they  persist  for  considerable  periods  in  sour 
milk,  butter,  and  cheese.  Milk  may  be  examined  for  the 
presence  of  tubercle  bacilli  by  microscopical  methods  and  by 
the  inoculation  of  experimental  animals. 

Microscopical  examination  for  tubercle  bacilli.  The  organ- 
ism of  tuberculosis  cannot  be  stained  by  the  use  of  ordinary 
stains.  A  solution  having  an  intense  staining  power  must  be 
employed.  The  cells  once  stained,  retain  the  color  even  when 
treated  with  dilute  mineral  acids.  Thus  a  preparation  may 
be  stained,  the  color  removed  from  all  the  material  except 
the  tubercle  bacilli,  and  then  the  preparation  restained  with 
a  contrast  stain. 

The  microscopical  examination  of  milk  for  tubercle  bacilli 
is  not  satisfactory,  since  these  are  likely  to  be  present  in 
such  small  numbers  as  to  be  very  difficult  or  impossible 
to  find. 

Procedure.  The  milk  should  be  heated  to  70°  C.  for  a  few 
moments  to  break  up  the  clusters  of  fat  globules  and  prevent, 
as  far  as  possible,  the  bacteria  being  carried  with  the  cream 
in  the  subsequent  process  of  centrif ugalization.  Ten  to  fifteen 


MILK  HYGIENE  113 

cubic  centimeters  of  the  milk  are  centrifugalized  for  twenty 
minutes  at  a  speed  of  2000  revolutions  per  minute.  The 
supernatant  liquid  is  removed  by  a  fine-pointed  glass  tube 
attached  to  an  aspirating  pump  by  means  of  a  rubber  tube. 
The  cream  adhering  to  the  sides  of  the  tube  is  removed  by 
the  use  of  absorbent  cotton.  Smears  are  prepared  from  the 
sediment,  dried,  fixed,  and  stained  as  follows:  The  preparation 
is  flooded  with  carbol-fuchsin  and  heated  over  a  water  bath 
or  by  passing  through  the  flame  of  a  Bunsen  burner  until  the 
preparation  steams.  Allow  the  hot  dye  to  act  three  to  four 
minutes,  and  then  wash.  Decolorize  with  a  5  per  cent  solu- 
tion of  nitric  acid  in  80  per  cent  alcohol  until  the  red  color 
is  discharged.  Wash  and  stain  with  aqueous  methylene  blue 
for  a  minute.  Tubercle  bacilli  appear  as  slender  red  rods  on 
a  blue  field. 

Experience  should  be  gained  in  the  demonstration  of 
tubercle  bacilli  by  the  staining  and  examination  of  prep- 
arations from  sputa.  The  student  should  bear  in  mind  that 
the  tubercle  organism  in  sputum  is  longer  and  more  slender 
than  in  milk.  In  sputum  it  also  has  a  distinct  tendency  to 
form  a  "  beaded  "  appearance. 

In  manure,  and  in  dust  from  hay  and  fodder,  there  are  fre- 
quently found  organisms,  the  so-called  "acid-fast"  bacteria, 
which  possess  the  same  relation  to  decolorizing  agents  as  the 
tubercle  bacillus.  Many  of  these  are  very  similar  in  morphol- 
ogy to  the  tubercle  bacillus,  differing,  however,  in  cultural 
characters  and  in  pathogenicity.  Through  the  contamination 
of  the  milk  with  manure  and  barn  dust  this  type  of  bacteria 
is  likely  to  find  its  way  into  the  milk,  and  care  must  be  exer- 
cised in  the  examination  of  milk  sediments  not  to  mistake 
such  forms  for  true  tubercle  bacilli.  A  strict  differentiation 
in  such  cases  can  only  be  made  by 'animal  inoculation. 


114         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Examination  for  tubercle  bacilli  by  animal  inoculation. 

The  inoculation  of  animals  susceptible  to  tuberculosis  is  a 
much  more  delicate  test  for  the  presence  of  tubercle  bacilli 
in  milk  than  the  microscopical  examination  of  the  sediment. 
Highly  virulent  milk  from  a  tuberculous  animal  may  be  di- 
luted as  much  as  a  million  times,  and  the  presence  of  tuber- 
cle bacilli  in  the  mixed  milk  may  be  readily  demonstrated  by 
animal  inoculation.  The  guinea  pig  is  used,  being  the  most 
susceptible  of  the  experimental  animals  to  artificial  inocula- 
tion, and  very  resistant  to  infection  in  a  natural  way. 

1.  The  unheated  milk  may  be  centrifugalized  as  for  the 
microscopical  examination.  Five  cubic  centimeters  of  the 
mixed  cream  and  sediment  are  injected  into  the  abdominal 
cavity.  The  syringe  is  filled  with  the  mixture,  the  skin  and 
abdominal  wall  of  the  animal  picked  up  between  the  thumb 
and  finger,  and  the  needle  of  the  syringe  thrust  through  the 
skin  and  wall  at  right  angles  to  the  body.  The  inoculation 
should  be  made  toward  the  side  rather  than  in  the  median  line 
of  the  abdomen.  Frequently  the  animals  die  in  a  short  time 
of  acute  peritonitis,  caused  by  other  organisms  present  in  the 
milk  or  on  account  of  a  puncture  of  the  intestinal  wall  in  the 
inoculation  process.  The  animals  which  survive  should  be 
chloroformed  in  sixty  or  seventy-five  days,  and  a  careful  post- 
mortem examination  made.  The  point  of  inoculation  should 
be  examined,  as  well  as  the  liver,  spleen,  kidneys,  peritoneum 
and  membranes  of  the  abdominal  cavity,  and  the  lymph  glands, 
especially  of  the  inguinal  region.  The  condition  of  the  lungs 
should  also  be  noted.  If  lesions  are  found,  the  results  must 
be  controlled  by  a  microscopical  examination  of  the  tissue  in 
smear  preparations,  and  in  suspicious  cases  by  a  second  inocu- 
lation of  animals  with  a  suspension  of  the  suspected  tissues 
of  the  first  macerated  in  a  mortar  with  sterile  water.  The 


MILK  IIYGIKM:  115 

suspension  may  be  allowed  to  settle  or  filtered  through  a  plug 
of  glass  wool,  in  order  to  prevent  stopping  of  the  needle  of  the 
syringe  with  bits  of  tissue. 

These  precautionary  measures  must  be  taken,  for  the  in- 
jection of  large  quantities  of  butter  fat  into  the  abdominal 
cavity  of  guinea  pigs  often  produces  tubercles  that  are  very 
similar  to  those  produced  by  the  tubercle  organism.  Inocu- 
lation of  a  second  animal  with  such  tissue  is  without  effect. 

2.  A  more  rapid  method,  and  one  whose  delicacy  is  as  great 
as  the  intraperitoneal  injection  of  a  mixed  cream  and  sedi- 
ment, is  the  injection  of  1  cc.  of  the  whole  milk  into  the 
muscles  of  the  thigh  of  a  guinea  pig.  The  enlargement  of  the 
lymph  glands  in  the  inguinal  region  is  evidence  of  the  pres- 
ence of  tubercle  bacilli.  The  changes  often  appear  as  early  as 
the  twelfth  day  after  inoculation.  For  more  detailed  methods 
of  animal  inoculation  and  post-mortem  examination,  the  stu- 
dent is  referred  to  standard  works  on  medical  bacteriology. 

Exercise.  A  demonstration  of  the  inoculation  and  post-mortem 
examination  of  an  animal  should  be  made  by  the  instructor. 

Examination  for  pyogenic  organisms.  The  pyogenic  or- 
ganism sought  for  in  milk  is  Streptococcus  pyogenes,  since  this 
is  the  most  virulent  of  all  the  pus-forming  bacteria,  and  the 
most  frequently  found  in  outbreaks  of  contagious  garget.  The 
examination  is  of  little  value,  except  in  the  case  of  perfectly 
fresh  market  milk,  or  in  high-grade  milk  whose  bacterial  con- 
tent is  largely  from  the  interior  of  the  udeter.  The  pyogenic 
organisms  do  not  find  favorable  conditions  for  growth  in 
market  milk,  and  are  soon  crowded  out  by  lactic  organisms ; 
hence  in  milk  of  high  germ  content  their  detection  is  difficult. 

The  streptococci  find  most  favorable  conditions  for  growth 
on  lactose  agar  at  37°  C.  Suspicious  colonies  on  such  plates 
should  be  inoculated  into  lactose  broth,  and  this  examined 


116          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

after  twenty-four  hours'  incubation  for  chains  of  cocci,  by 
preparing  stained  preparations. 

The  student  should  become  familiar  with  the  morphologi- 
cal, cultural,  and  physiological  characteristics  of  Streptococcus 
pyogencs  and  Staplilococcus  pyogenes  aureus,  the  common  yel- 
low pus  organism. 

Microscopical  examination  of  milk.  In  the  milk  of  all 
cows  is  to  be  found  a  greater  or  less  number  of  cellular  ele- 
ments, such  as  colostrum  cells,  epithelial  cells,  and  white 
blood  corpuscles.  Whenever  there  is  any  inflammation  in 
the  udder,  whether  the  cause  is  the  presence  of  pyogenic  bac- 
teria or  a  physiological  disturbance  such  as  the  influence  of 
cold,  bruising,  etc.,  the  number  of  leucocytes  is  greatly  in- 
creased. Considerable  significance  is  attached  to  the  pres- 
ence of  large  numbers  of  these  cells.  The  milk  drawn  from 
perfectly  healthy  animals  varies  so  widely  in  content  of 
leucocytes  that  it  is  difficult  to  establish  a  standard  and  to 
determine  what  significance  they  possess.  The  milk  of  nor- 
mal, healthy  cows  may  contain  from  a  few  hundred  leucocytes 
to  several  million  per  cubic  centimeter.  In  many  cities  a  limit 
of  500,000  leucocytes  per  cubic  centimeter  has  been  adopted. 
Milk  containing  a  greater  number  than  this  is  looked  upon  as 
containing  pus.  While  the  milk  of  individual  animals  in  a 
perfect  state  of  health  may  frequently  exceed  this  number, 
it  is  certain  that  the  mixed  milk  from  a  herd  of  considerable 
size  (twelve  animals  or  more)  will  not  show  a  higher  number 
unless  one  or  more  animals  have  udder  trouble.  Standards 
of  this  character  should,  however,  be  regarded  as  suggestive 
rather  than  as  final.  Where  negative  findings  are  determined 
they  may  be  taken  as  indicating  a  normal,  healthy  condition  ; 
where  results  show  an  excess  of  leucocytes,  it  should  lead  to 
a  careful  physical  examination  of  the  herd. 


MILK  HYGIENE  117 

Two  methods  are  used  for  determining  the  number  of 
leucocytes  in  milk. 

Examination  for  leucocytes :  Doane-Buckley  method.  Cen- 
trifuge tubes,  which  are  graduated  at  0.5  cc.  and  10  cc.,  to 
aid  in  filling  and  in  the  subsequent  removal  of  the  supernatant 
liquid,  are  employed.  These  are  filled  with  the  milk  to  be 
examined.  They  are  then  heated  to  70°-75°  C.  for  five  to 
ten  minutes,  and  well  shaken  after  heating.  The  fat  in  milk 
is  largely  in  aggregates  of  globules,  which  seem  to  frequently 
inclose  leucocytes.  If  the  milk  is  heated,  the  aggregates  of  fat 
globules  are  broken  up  and  the  leucocytes  freed.  If  the  un- 
heated  milk  is  used,  a  large  number  of  leucocytes  are  to  be 
found  in  the  cream.  Since  only  the  sediment  can  be  exam- 
ined, the  leucocytes  must  be  concentrated  in  it. 

The  tubes  of  warm  milk  are  centrifugalized  in  any  of  the 
ordinary  types  of  laboratory  centrifuges,  or  a  Babcock  tester 
may  be  used.  Unless  a  high-speed  centrifuge  is  available,  it 
is  difficult  to  remove  the  smaller  fat  globules.  If  considerable 
numbers  are  left,  they  seriously  interfere .  with  the  counting 
of  the  leucocytes.  These  small  fat  globules  may  be  removed 
from  the  milk  by  the  use  of  high-speed  machines,  or  by  cen- 
trifugalizing  the  milk  for  eight  minutes  at  2000  revolutions 
per  minute,  removing  the  upper  layers  as  described  below, 
filling  with  distilled  water,  and  centrifugalizing  again  for 
three  or  four  minutes. 

The  cream  and  milk  are  best  removed  by  the  use  of  a 
fine-pointed  glass  tube  of  2  mm.  internal  diameter,  attached 
to  an  aspirating  pump  by  thick-walled  rubber  tubing.  It  is 
advisable  to  place  between  the  pump  and  glass  tube  a  bottle 
to  receive  the  milk.  By  touching  the  point  of  the  tube  to  the 
liquid  the  very  upper  layers  may  be  removed.  The  fat  that 
adheres  to  the  wall  of  the  centrifuge  tube  should  be  wiped 


118          EXPERIMENTAL   DAIRY  BACTERIOLOGY 


off  with  absorbent  cotton.  The  aspirating  is  continued  until 
but  J-  cc.  of  milk  remains.  The  sediment  is  well  mixed  in 
the  liquid  by  means  of  a  glass  rod. 

The  time  of  centrifugalization  must  be  sufficient  to  cause 
as  complete  a  separation  of  the  fat  as  possible,  since  large 
numbers  of  fat  globules  interfere  seriously  with  the  counting, 

since  they  rise  to  the 
top  of  the  cell.  A 
speed  and  time  neces- 
sary to  remove  the 
fat  will  be  sufficient 
to  throw  down  all  leu- 
cocytes. 

A  T  h  o  m  a-Z  e  i  s  s 
blood-corpuscle-count- 
ing cell  is  used.  This 
is  formed  of  a  heavy 
glass  slide  on  which  is 
cemented  a  square 
piece  of  glass  having 
a  circular  hole  in  the 
center.  In  the  center 
of  this  hole  is  cemented 
011  the  slide  a  piece  of 

glass  bearing  on  its  upper  surface  a  ruled  area  made  up  of 
400  squares.  The  entire  ruled  area  comprises  1  sq.  mm.  The 
thickness  of  the  glass  ring  is  0. 1  mm.  greater  than  that  of  the 
glass  bearing  the  ruled  area.  Thus,  when  a  perfectly  plane 
piece  of  glass  is  used  as  a  cover,  the  depth  of  the  cell  over 
the  ruled  area  is  0.1  mm.;  thus  the  volume  of  liquid  over 
the  rulings  is  equal  to  0.1  cu.  mm.  Each  of  the  400  squares 
represents  a  volume  of  1/4000  cu.  mm. 


FIG.  17.   THE  RULED  AREA  OF  THE  TIIOMA- 
ZEISS  BLOOD  COUNTER 

The  area  of  all  the  squares  is  1  sq.  mm.   The 

four  hundred  squares  are  separated  into  groups 

of  sixteen  by  the  double  lines 


MILK  HYGIENE 


119 


With  the  rod  a  drop  of  thoroughly  mixed  milk  and  sedi- 
ment is  transferred  to  the  cell  and  the  cover  placed  in  position. 
The  transfer  should  be  made  at  once  after  mixing,  and  the 
drop  should  be  just  large  enough  to  fill  the  cell  completely, 
or  at  least  three  fourths  of  it,  but  should  not  overflow  into 
the  moat.  The  preparation  should  be  allowed  to  stand  a 


FIG.  18.   THOMA-ZEISS  BLOOD-CORPUSCLE-COUNTING  CELL 

A,  plain  glass  cover;  /?,  glass  with  circular  hole  in  its  center;  C,  glass  bear- 
ing ruled  area.   The  distance  from  the  upper  surface  of  C  to  the  lower  surface 
of  A  is  0.1  mm. 

moment  after  filling  and  covering,  to  allow  the  fat  globules 
to  rise  to  the  upper  portion  of  the  cell  and  the  leucocytes 
to  settle. 

The  counting  is  done  with  a  £-inch  objective  of  long  work- 
ing distance  (0.66  numerical  aperture),  counting  the  number 
of  leucocytes  in  the  entire  ruled  area  in  milks  low  in  con- 
tent. With  increased  content  a  less  number  of  squares  may 
be  counted.  The  small  squares  will  be  found  divided  by  double 
lines  into  sets  of  sixteen,  in  order  to  facilitate  the  counting. 
At  least  six  of  these  sets  should  be  counted.  The  average 
number  of  leucocytes  per  small  square  multiplied  by  200,000 


120          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

gives  the  number  per  cubic  centimeter  in  the  original  milk. 
The  factor  200,000  is  obtained  as  follows :  The  leucocytes 
are  concentrated  in  1/20  part  of  the  milk.  In  order  to  de- 
termine the  number  in  each  cubic  centimeter  of  the  milk,  the 
number  found  in  1/4000  cu.  mm.  of  the  liquid  examined  must 

be  multiplied  by  -— ^ ,  or  200,000.  ' 

The  student  should  become  familiar  with  the  appearance 
of  white  blood  corpuscles  by  the  examination  of  blood.  Blood 
is  easily  obtained  by  winding  a  rubber  band  about  one  finger, 
and  when  the  veins  are  gorged  with  blood,  pricking  oneself  at 
the  base  of  the  nail  with  a  glass  rod  drawn  out  to  a  fine  point. 
The  puncture  should  be  made  by  a  quick  stroke.  The  drop  of 
blood  is  mixed  with  nine  volumes  of  0.3  per  cent  glacial  acetic 
acid  and  examined  in  the  blood  counter.  The  red  corpuscles 
will  be  scarcely  visible  in  this  form  of  preparation. 

Examination  for  leucocytes ;  smeared-sediment  method. 
Small  glass  tubes  closed  at  either  end  with  soft  rubber  stop- 
pers are  used.  The  tubes  should  be  of  6  mm.  internal  diam- 
eter and  of  sufficient  length  to  hold  2  cc.  When  filled  with 
milk,  which  has  been  heated  as  in  the  Doane-Buckley  method, 
they  may  be  centrifugalized  in  any  form  of  centrifuge.  A  flat 
aluminum  disk,  with  upturned  edge,  may  be  attached  to  an 
ordinary  centrifuge.  The  disk  is  provided  with  appropriate 
clamps  for  holding  the  small  tubes  in  position,  and  with  a 
cover  held  in  place  by  a  screw.  With  this  form  of  centri- 
fuge head  much  greater  speed  can  be  attained,  since  the  air 
resistance  is  much  less  than  with  the  ordinary  tubes.  A 
source  of  power  that  would  produce  1200-1500  revolutions 
per  minute  with  the  ordinary  head  will  produce  3000  with 
the  disk..  Twenty  samples  may  be  centrifugalized  at  once 
in  an  apparatus  of  this  kind. 


MILK  HYGIENE  121 

After  centrifugalization  the  stopper  at  the  cream  end  is 
removed  and  the  layer  of  cream  disturbed  with  a  needle,  the 
milk  and  cream  poured  out,  and,  without  placing  the  tube 
right  side  up,  the  lower  stopper  with  the  adhering  sediment 
is  removed.  The  sediment  is  smeared  over  a  definite  area, 
4  sq.  cm.  on  an  ordinary  glass  slide,  by  use  of  the  stopper. 
The  smear  should  be  as  uniform  in  distribution  as  possible. 
The  use  of  a  drop  of  water  will  aid  in  this  respect.  The  smear 
is  allowed  to  air  dry,  and  is  then  stained  with  methylene  blue 
for  a  few  moments.  The  stain  is  carefully  washed  off  so  as 
not  to  loosen  the  smear,  and  the  slide  allowed  to  dry. 

It  is  examined  with  an  oil-immersion  lens,  placing  the  oil 
directly  on  the  dry  smear.  The  average  number  of  leucocytes 
per  field  is  determined  by  counting  a  number  of  fields.  The 
number  that  must  be  counted  depends  upon  the  evenness  of 
distribution  of  -the  sediment.  At  least  fifty  should  be  ex- 
amined. The  diameter  of  the  field  of  the  oil-immersion  lens 
is  determined  with  a  stage  micrometer.  The  number  of  mi- 
croscopic fields  in  the  total  area  of  the  smear  is  calculated, 
and  from  the  data  thus  obtained  the  average  number  of  leu- 
cocytes per  cubic  centimeter  of  the  milk  examined  is  deter- 
mined. The  student  should  prepare  preparations  of  blood, 
staining  them  with  methylene  blue,  so  as  to  become  familiar 
with  the  appearance  of  the  white  corpuscles  when  stained. 
The  blood  is  obtained  as  previously  described.  A  drop  of  blood 
is  placed  on  a  clean  slide  and  spread  by  means  of  another 
slide  whose  corners  at  one  end  have  been  chipped  off.  This  is 
brought  in  contact  with  the  drop  of  blood  and  drawn  along 
the  slide,  holding  the  spreader  at  an  angle  of  45°.  A  thin 
film  of  blood  is  thus  deposited  along  the  slide,  the  broken 
corners  preventing  the  film  extending  to  the  edges  of  the 
slide. 


122         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Exercise.  Each  student  will  examine  samples  of  milk  for  leucocytes 
from  healthy  animals,  and,  if  possible,  from  animals  having  garget. 
Both  methods  should  be  employed  on  the  same  sample,  if  possible. 
Compare  results  obtained  by  the  different  methods. 

Examination  for  fibrin.  In  inflammatory  troubles  of  the 
udder  white  blood  corpuscles  are  not  the  only  constituent  of 
the  blood  passing  from  the  blood  vessels  into  the  glandular 
tissue  and  milk  ducts.  The  blood  serum,  with  its  dissolved 
constituents,  serum  albumen  and  fibrinogen,  passes  out  to  a 
greater  or  less  extent.  Under  appropriate  conditions  the  fibrin- 
ogen  is  changed  to  the  insoluble  fibrin,  which,  when  present 
in  any  considerable  amounts,  can  be  detected  by  microscopic 
examination.  The  fibrin  determines  the  distribution  of  the 
leucocytes.  When  fibrin  is  present  in  small  quantities  or  not 
at  all,  the  leucocytes  are  evenly  distributed.  In  the  presence 
of  fibrin  larger  or  smaller  clumps  are  to  be  found. 

The  milk  may  be  centrifugalized,  as  in  the  smeared-sedi- 
ment  method  for  leucocytes.  The  sediment  should  be  spread 
on  a  slide,  noting  whether  any  dough-like  material  is  present. 
The  smear  is  stained  as  follows,  after  being  allowed  to  thor- 
oughly air  dry  :  Flood  with  aniline-water  gentian  violet  (p.  43). 
Allow  stain  to  remain  on  the  preparation  for  five  minutes,  and 
then  wash.  Flood  with  iodine  solution  (p.  43),  wash  off  after 
one  or  two  minutes,  and  add  aniline  oil  from  time  to  time 
until  the  color  is  completely  discharged,  as  shown  by  the  color- 
less condition  of  the  oil.  Wash  in  water,  dry,  and  examine. 
The  fibrin  should  appear  as  threads  stained  dark  purple. 

Exercise.  The  student  should  familiarize  himself  with  the  appear- 
ance of  fibrin  in  blood.  Examine  normal  milk  for  fibrin ;  also  that 
from  an  animal  having  garget. 

Direct  enumeration  of  bacteria  in  milk.  In  control  work, 
such  as  must  be  carried  on  in  the  municipal  laboratories  of 


MILK  HYGIENE  123 

cities  which  have  established  a  numerical  standard  in  refer- 
ence to  the  bacterial  content  of  milk,  it  is  desirable  to  have 
some  method  by  which  it  can  quickly  and  easily  be  deter- 
mined whether  or  not  a  sample  of  milk  exceeds  the  estab- 
lished standard.  The  analyst  does  not  care  to  know  the  exact 
number  of  organisms  present,  but  merely  whether  the  milk 
is  good,  fair,  or  poor,  with  reference  to  the  standard  adopted. 

The  smear  prepared  from  the  milk  for  determination  of 
the  number  of  leucocytes  present  will  serve  for  this  purpose. 
The  average  number  of  bacteria  per  field  of  the  oil-immersion 
lens  is  determined  by  counting  those  appearing  in  a  number 
of  fields.  From  the  average  per  field  and  the  areas  of  field 
and  smear  the  number  of  bacteria  per  cubic  centimeter  of 
milk  may  be  calculated.  Each  diplococcus  or  diplobacillus, 
each  chain  or  clump  of  organisms,  should  be  counted  as  one, 
since  such  aggregations  will  usually  produce  but  one  colony 
on  the  plate  cultures.  The  legal  standard  always  refers  to  the 
number  of  organisms  as  determined  by  some  method  of  plating. 

The  value  of  this  method  depends  much  on  the  experience 
of  the  analyst.  With  considerable  experience  it  is  certain 
that  in  many  cases  it  is  possible  to  determine  whether  a 
given  sample  falls  below  or  greatly  exceeds  the  standard. 
Standards  of  this  character,  however,  should  not  be  inter- 
preted too  strictly.  They  serve  as  admirable  aids  in  milk  con- 
trol, inasmuch  as  the  inspector  is  thus  able  to  concentrate 
his  attention  on  those  cases  which  are  designated  as  suspi- 
cious by  the  laboratory  analysis.  Where  direct  tests  show 
a  perfectly  normal  condition,  plating  is  unnecessary,  and 
only  the  questionable  samples  need  be  further  examined. 
The  exact  counting  is  not  necessary,  a  rapid  glance  over  the 
preparation  enabling  the  experienced  analyst  to  judge  of  the 
quality  of  the  milk.  The  examination  also  gives  the  analyst 


124         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

some  idea  as  to  the  dilutions  to  be  used  in  the  preparation 
of  plate  cultures,  so  as  to  obtain  plates  with  the  desired 
number  of  colonies. 

Microscopic  examination  for  streptococci.  As  has  been 
previously  pointed  out,  the  pyogenic  organisms  most  likely 
to  be  the  cause  of  udder  troubles  are  streptococci  of  the 
type  of  Streptococcus  pyogenes.  In  the  examination  of  the 
smears  prepared  from  the  milk  sediments,  the  form  of  organ- 
isms predominating  should  be  noted.  The  value  of  the  ex- 
amination depends  much  upon  the  age  of  the  milk.  It  is 
impossible  to  distinguish  certain  types  of  lactic-acid  organ- 
isms from  pyogenic  streptococci,  since  their  morphology  is  so 
nearly  identical.  Thus,  in  milk,  in  which  the  lactic  organ- 
isms have  increased  in  numbers,  such  an  examination  is  of 
doubtful-  value.  In  perfectly  fresh  milk  long  chains  of  strep- 
tococci are  indicative  of  udder  trouble.  In  milk  of  animals 
suffering  from  garget  immense  numbers  of  such  organisms 
may  frequently  be  found  in  the  sediment. 


APPENDIX  A 


PIPETTES 

Preparation  of  pipettes.  The  quantitative  bacteriological  analyses 
of  milk  and  other  dairy  products  involves  the  use  of  a  large  amount 
of  glassware,  especially  pipettes.  One-cc.  pipettes,  suitable  for  use 
in  quantitative  work,  can  be  easily  prepared.  Glass  tubing  of 
4  mm.  internal  diameter  is  cut  into  pieces  20  inches  long.  The  piece 
is  heated  in  the  center  and  drawn  out 
so  as  to  form  the  tips  of  the  pipettes. 
The  pieces  are  then  cut  in  two,  and 
both  ends  slightly  fused  in  the  flame 
to  remove  the  sharp  edges.  The  pi- 
pettes are  rendered  perfectly  free  from 
grease  and  dirt  by  boiling  them  in  a 
solution  of  washing  powder,  rinsing 
well  with  distilled  water,  and  heating 
in  a  hot-air  sterilizer  for  an  hour  at 
175°-200°  C.,  or  by  washing  in  alcohol 
and  then  in  ether. 

A  Schuster  dropping  bottle  is  thor- 
oughly cleaned  and  dried.  The  top  of 
the  bottle  should  be  drawn  out  so  it 
can  be  easily  introduced  within  the 
lumen  of  the  pipette,  and  the  hole  in 
the  bottle  made  very  small.  In  the 
bottle  are  placed  13.6  grams  of  perfectly  clean*,  dry  mercury.  The 
upper  part  of  each  pipette  is  coated  with  a  thin  coat  of  paraffin  by 
warming  the  glass  and  rubbing  it  on  a  piece  of  paraffin.  The  pipette 
is  held  between  the  first  two  fingers,  the  tip  resting  on  the  ball  of 
the  thumb.  The  mercury  is  poured  into  the  pipette  slowly  so  as  to 
allow  the  air  to  escape.  A  very  small  bubble  of  air  will  usually  be 
retained  in  the  tip,  but  this  may  be  neglected.  The  upper  level  of 
the  mercury  which  indicates  a  volume  of  one  cc.  is  marked  on  the 
paraffin  by  a  pin  scratch.  The  mercury  is  returned  to  the  dropping 

125 


FIG.  19.   SCHUSTER  DROPPING 
BOTTLE 

The  top  of  the  bottle  is  drawn  so 

as  to  form  a  capillary  tube  from 

which  the  mercury  will  run 

slowly 


126          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

bottle  by  allowing  it  to  run  from  the  top  of  the  pipette,  and  the 
operation  repeated  with  each  pipette.  If  the  glassware  is  free  from 
moisture  and  grease,  and  the  mercury  clean,  there  should  be  no 
adherence  of  the  mercury  to  the  glass  and  no  tendency  for  it  to  form 
small  globules.  A  permanent  mark  is  placed  on  the  pipettes  by 
scratching  through  the  paraffin  with  a  fine  needle  and  filling  the 
scratch  with  hydrofluoric  acid  or  diamond  ink  and  allowing  the  same 
to  act  for  five  minutes  ;  then  wash  off,  remove  paraffin  by  warming 
sufficiently  to  melt  it,  and  wipe  with  paper.  A  mark  may  be  made 
by  the  use  of  a  sharp  file,  which  method  avoids  the  use  of  paraffin 
and  hydrofluoric  acid. 


APPENDIX  B 

DESCRIPTIVE  CHART,  SOCIETY  OF  AMERICAN 
BACTERIOLOGISTS 

PREPARED  BY  F.  I).  CHESTER,  F.  P.  GOKIIAM,  ERWIN  F.  SMITH,  COMMIT- 
TEE ON  METHODS  OF  IDENTIFICATION  OF  BACTERIAL  SPECIES 

DETAILED  FEATURES 

NOTE.   Underscore  required  terms. 

I.  MORPHOLOGY 

1.  Vegetative  cells,  medium  used 

temp ,  age ,  days 

Form,  round,  short  rods,  long  rods,  short  chains,  long  chains,  jila- 
iii'  nts,  commas,  short  spirals,  long  spirals,  clostridium,  cuneale, 
clavate,  curved. 

Limits  of  size 

Size  of  majority 

Ends,  rounded,  truncate,  concave. 

f  Orientation  (grouping) 

Agar  hanging    I  Chains  (number  of  elements) 

block  I  Short  chains,  long  chains. 

[  Orientation  of  chains,  parallel,  irregular. 

2.  Sporangia,  medium  used.., ,  temp 

age ,  days 

Form,  elliptical,  short  rods,  spindled,  clavate,  drumsticks. 
Limits  of  size Size  of  majority 

f  Orientation  (grouping).... 
Agar  hanging 

block  ^  Chains  (number  of  elements).... 

[  Orientation  of  chains,  parallel,  irregular. 
Location  of  endospores,  central,  polar. 

3.  Endospores. 

Form,  round,  elliptical, 
Limits  of  size 
Size  of  majority 

127 


128         EXPERIMENTAL  DAIRY  BACTERIOLOGY 

Wall,  thick,  thin. 

Sporangium  wall,  adherent,  not  adherent. 

Germination,  equatorial,  oblique,  polar,  bipolar,  by  stretching. 

4.  Flagella  No. Attachment,  polar,  bipolar,  peritrichiate. 

How  stained 

5.  Capsules,  present  on 

6.  Zoogloea,  Pseudozoogloaa. 

7.  Involution  forms,  on in days  at °  C. 

8.  Staining  reactions. 

1  :  10  watery  fuchsin,  gentian  vio!et,  carbol-f  iichsin,  Loeffler's 

alkaline  methylene  blue. 
Special  stains. 

Gram Glycogen 

Fat Acid  fast 

Neisser 

II.    CULTURAL  FEATURES 

1.  Agar  stroke. 

Growth,  invisible,  scanty,  moderate,  abundant. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose, 

arborescent,  rhizoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Luster,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 
Optical  characters,  opaque,  translucent,  opalescent,  iridescent. 

Chromogenesis  (5) 

Odor,  absent,  decided,  resembling 

Consistency,    slimy,   butyrous,  viscid,   membranous,    coriaceous, 

brittle. 
Medium,  grayed,  browned,  reddened,  blued,  greened. 

2.  Potato. 

Growth,  scanty,  moderate,  abundant,  transient,  persistent. 
Form  of  growth,  filiform,  echinulate,  beaded,  spreading, plumose, 

arborescent,  rhizoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Luster,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 
Chromogenesis  (5) Pigment  in  water, 

insoluble,  soluble  ;  other  solvents 

Odor,  absent,  decided,  resembling 


APPENDIX  B  129 

Consistency ,  slimy,  bnfi/rons,  r/.sr/V/,  membranous,  coriaceous,  brittle. 
Medium,  f/r<ii/i-</,  hrowned,  reddened,  blued,  greened. 

3.  LoefQer's  blood  serum. 

Stroke,  invisible,  scant)/,  moderate,  abundant. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose, 

arborescent,  rhizoid. 

Elevation  of  growth,  fiat,  effuse,  raised,  convex. 
Luster,  (/listening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

Chromogenesis  (5) 

Medium,  grayed,  browned,  reddened,  blued,  greened. 
Liquefaction  begins  in d.,  complete  in d. 

4.  Agar  stab. 

Growth,  uniform,  best  at  top,  best  at  bottom;  surface  growth 
scanty,  abundant :  restricted,  widespread. 

Line  of  puncture,  filiform,  beaded,  papillate,  villous,  plumose,  ar- 
borescent ;  liquefaction. 

5.  Gelatin  stab. 

Growth,  uniform,  best  at  top,  best  at  bottom. 

Line  of  puncture,  filiform,  beaded,  papillate,   villous ,  plumose, 

arborescent. 
Liquefaction,   crateriform,   napiform,  infundibuliform,   saccate, 

stratiform  ;  begins  in d.,  complete  in d. 

Medium,  fluorescent,  browned 

6.  Nutrient  broth. 

Surface  growth,  ring,  pellicle,  fiocculent,  membranous,  none. 
Clouding,  slight,  moderate,  strong;   transient,  persistent;   none; 

fluid  turbid. 

Odor,  absent,  decided,  resembling 

Sediment,  compact,  fiocculent,  granular,  flaky,  viscid  on  agitation, 

abundant,  scant. 

7.  Milk. 

Clearing  without  coagulation. 
Coagulation,  prompt,  delayed,  absent. 

Extrusion  of  whey,  begins  in d. 

Coagulum,  slowly  peptonized,  rapidly  peptonized. 

Peptonization  begins  on d.,  complete  on d. 

Reaction,  Id ,  2d ,  4d :,  10 d ,  20 d.... 

Consistency,  slimy,  I'iscid,  unchanged. 
Medium,  browned,  reddened,  blued,  greened. 
Lab  ferment,  present,  absent. 


130          EXPERIMENTAL  DAIRY  BACTERIOLOGY 

8.  Litmus  milk. 

Acid,  alkaline,  acid  then  alkaline,  no  change. 

Prompt  reduction,  no  reduction,  partial  slow  reduction. 

9.  Gelatin  colonies. 

Growth,  slow,  rapid. 

Form,  punctiform,  round,  irregular,  amoeboid,  mycelioid,  filamen- 
tous,  rhizoid. 

Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  crateriform, 
(liquefying). 

Edge,  entire,  undulate,  lobate,  erose,  lacerate,  Jimbriate,  filamen- 
tous, floccose ,  curled. 

Liquefaction,  cup,  saucer,  spreading. 

10.  Agar  colonies. 

Growth,  slow,  rapid  (temperature '. ). 

Form,  punctiform,  round,  irregular,  amoeboid,  mycdioid ,  filamen- 
tous, rhizoid. 

Surface,  smooth,  rough,  concentrically  ringed,  radiate,  striate. 

Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  umbonate. 

Edge,  entire,  undulate,  lobate,  erose,  lacerate,  fimbriate,  Jloccose, 
curled. 

Internal  structure,  a morphous,  finely  granular,  coarsely  (jranular, 
grumose,  filamentous,  Jloccose,  curled. 

11.  Starch  jelly. 

Growth,  scanty,  copious. 

Diastasic  action,  absent,  feeble,  profound. 

Medium  stained 

12.  Silicate  jelly  (Fermi's  solution). 

Growth,  copious,  scanty,  absent. 

Medium  stained 

13.  Conn's  solution. 

Growth,  copious,  scanty,  absent. 
Medium,  fluorescent,  nonftuorescent. 

14.  Uschinsky's  solution. 

Growth,  copious,  scanty,  absent. 
Fluid,  viscid,  not  viscid. 

15.  Sodium  chloride  in  bouillon. 

Per  cent  inhibiting  growth 

16.  Growth  in  bouillon  over  chloroform,  unrestrained ,  feeble ,  absent. 

17.  Nitrogen. 

Obtained  from  peptone,  asparagin,  glycocoll,  urea,  ammonia  salts, 
nitrogen. 


APPENDIX  B 

18.  Best  media  for  long-continued  growth  . 

19.  Quick  tests  for  differential  purposes 


131 


HI.  PHYSICAL  AND  BIOCHEMICAL   FEATURES 


1.  Fermentation  tubes  con- 
taining peptone  water  or 
sugar-free  bouillon  and 


Gas  production,  in  per  cent 


Growth  in  closed  arm 


Amount  of  acid  produced  in  1  d. 


Amount  of  acid  produced  in  2  d. 


Amount  of  acid  produced  in  4  d. 


2.  Ammonia    production,  feeble,  moderate,  strong,  absent,  masked  by 

acids. 

3.  Nitrates  in  nitrate  broth,  reduced,  not  reduced. 

Presence  of  nitrites ,  ammonia 

"        "  nitrates ,  free  nitrogen 

4.  Indol  production,  feeble,  moderate,  strong. 

5.  Toleration  of  acids,  great,  medium,  slight. 

Acids  tested 

6.  Toleration  of  NaOH,  great,  medium,  slight. 

7.  Optimum   reaction  for  growth  in  bouillon,  stated  in  terms  of 

Fuller's  scale 

8.  Vitality  on  culture  media,  brief,  moderate,  long. 

9.  Temperature  relations : 

Thermal  death  point  (10  minutes'  exposure  in  nutrient  broth 
when  this  is  adapted  to  growth  of  organism) C. 

Optimum  temperature  for  growth C.,  or  best  growth 

at  15°  C.,  20° C.,  25°  C.,  30°  C.,  37°  C.,  40°  C.,  50° C.,  60°  C. 


132         EXPERIMENTAL  DAIRY  BACTERIOLOGY 


Maximum  temperature  for  growth C. 

Minimum  temperature  for  growth C. 

10.  Killed  readily  by  drying  :  resistant  to  drying. 

11.  Per  cent  killed  by  freezing  (salt  and  crushed  ice  or  liquid  air) 


12.  Sunlight :  exposure  on  ice  in  thinly  sown  agar  plates  :  one  half 

plate  covered  (time  15  minutes),  sensitive,  not  sensitive. 
Per  cent  killed 

13.  Acids  produced 

14.  Alkalies  produced 

15.  Alcohols 

16.  Ferments  :  pepsin,  trypsin,  diastase,  invertase,  pectase,  cytase,  tyros- 

inase,  oxidase,  peroxidase,  Upase,  catalase,  glucase,  yalactase, 
lab,  etc 

17.  Crystals  formed 

18.  Effect  of  germicides. 


SUBSTANCE 

METHOD  USED 

MINUTES 

TEMPERATURE 

KILLING  QUANTITY 

AMOUNT  REQUIRED 
TO  RESTRAIN 
GROWTH 

APPENDIX  B 


133 


IV.   PATHOGENICITY 

1.  Pathogenic  to  animals. 

Insects,  crustaceans,  jishes,  reptiles,  birds,  mice,  rats,  guinea  pigs, 
rabbits,  dogs,  cats,  sheep,  goats,  cattle,  horses,  monkeys,  man. 

2.  Pathogenic  to  plants. 


3.  Toxins,  soluble,  endotoxins. 

4.  Nontoxin  forming. 

f>.  Immunity  bactericidal. 
0.  Immunity  nonbactericidal. 

7.  Loss  of  virulence  on  culture  media  :  prompt,  gradual,  not  observed 
in months. 

BRIEF  CHARACTERIZATION 

Mark  +  or  O,  and  when  two  terms  occur  on  a  line  erase  the  one  which 
does  not  apply  unless  both  apply 


MORPHOLOGY 

Diameter  over  1/x 

Chains,  filaments 

Endospores 

Capsules 

Zoogloea.   Pseudozooglcea 

Motile 

Involution  forms 

Gram's  stain 


CULTURAL  FEATURES 

BROTH 

Cloudy,  turbid 

Ring 

Pellicle 

Sediment 

AQAR 

Shining 

Dull 

Wrinkled 

Chromogenic 

134          EXPERIMENTAL  DAIRY  BACTERIOLOGY 


BRIEF  CHARACTERIZATION  (continued) 


CULTURAL  FEATURES  (continued) 

GELATIN 

PLATE 

Round 

Proteus-like 

Rhizoid 

Filamentous 

Curled 

GELATIN 

STAB 

Surface  growth 

Needle  growth 

POTATO 

Moderate,  absent 

Abundant 

Discolored 

Starch  destroyed 

Grows  at  37°  C. 

Grows  in  Cohn's  solution 

Grows  in  Uschinsky's  solution 

[  BIOCHEMICAL  FEATURES 

LIQUE- 
FACTION 

Gelatin  (2) 

Blood  serum 

Casein 

Agar,  mannan                                     . 

MILK 

Acid  curd 

Rennet  curd 

Casein  peptonized 

Indol  (3) 

Hydrogen  sulphide 

Ammonia  (3) 

Nitrates  reduced  (3) 

Fluorescent 

Luminous 

APPENDIX  B  135 

BRIEF  CHARACTERIZATION  (continued) 


DISTRIBUTION 

Animal  pathogen,  epizoon 

Plant  pathogen,  epiphyte 

Soil 

Milk- 

Fresh  water 

Salt  water 

Sewage 

Iron  bacterium 

Sulphur  bacterium 

NOTES 

(1)  For  decimal  system  of  group  numbers  see  Table  I.    This  will 
be  found  useful  as  a  quick  method  of  showing  close  relationships 
inside  the  genus,  but  is  not  a  sufficient  characterization  of  any 
organism. 

(2)  Gelatin  stab  cultures  shall  be  held  for  six  weeks  to  determine 
liquefaction. 

(3)  Ammonia  and  indol  tests  shall  be  made  at  the  end  of  the  tenth 
day,  nitrite  tests  at  the  end  of  the  fifth  day. 

(4)  Generic  nomenclature  shall  begin  with  the  year  1872  (Cohn's 
first  important  paper). 

Species  nomenclature  shall  begin  with  the  year  1880  (Koch's  dis- 
covery of  the  poured-plate  method  for  the  separation  of  organisms). 

(5)  Chromogenesis  shall  be  recorded  in  standard  color  terms. 


136 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


TABLE   I 

A  NUMERICAL  SYSTEM  OF  RECORDING  THE  SALIENT 
CHARACTERS  OF  AN  ORGANISM  (GROUP  NUMBER) 

100.  Endospores  produced 

200.  Endospores  not  produced 

10.  Aerobic  (strict) 

20.  Facultative  anaerobic 

30.  Anaerobic  (strict) 

1.  Gelatin  liquefied 

2.  Gelatin  not  liquefied 

0.1  Acid  and  gas  from  dextrose 

0.2  Acid  without  gas  from  dextrose 

0.3  No  acid  from  dextrose 

0.4  No  growth  with  dextrose 

.01  Acid  and  gas  from  lactose 

.02  Acid  without  gas  from  lactose 

.03  No  acid  from  lactose 

.04  No  growth  with  lactose 

.001  Acid  and  gas  from  saccharose 

.002  Acid  without  gas  from  saccharose 

.003  No  acid  from  saccharose 

.004  No  growth  with  saccharose 

.0001  Nitrates  reduced  with  evolution  of  gas 

.0002  Nitrates  not  reduced 

.0003  Nitrates  reduced  without  gas  formation 

.00001  Fluorescent 

.00002  Violet  chromogens 

.00003  Blue  chromogens 

.00004  Green  chromogens 

.00005  Yellow  chromogens 

.00006  Orange  chromogens 

.00007  Red  chromogens 

.00008  Brown  chromogens 

.00009  Pink  chromogens 

.00000  Nonchromogenic 

.000001  Diastasic  action  on  potato  starch  (strong) 

.000002  Diastasic  action  on  potato  starch  (feeble) 

.000003  Diastasic  action  on  potato  starch  (absent) 


APPENDIX  B  137 

.0000001     Acid  and  gas  from  glycerin 
.0000002     Acid  without  gas  from  glycerin 
.0000003     No  acid  from  glycerin 
.0000004     No  growth  with  glycerin 
The  genus  according  to  the  system  of  Migula  is  given 
its  proper  symbol,  which  precedes  the  number  thus:    (4) 

BACILLUS  COLI  (Esch.)  Mig.  becomes  B.       222.111102 

BACILLUS  ALCALIGENES  Petr.  "        B.       212.33:5 lo-j 

PSKIDOMONAS  CAMPESTRIS  (Pam.)  Sm.  "        Ps.      211.333151 

SUICIDA  Mig.  "        Bact.  222.232?03 


APPENDIX  C 

CONVERSION  FACTORS  FOE  THERMOMETER 

SCALES 

To  change  Centigrade  to  Fahrenheit :   (C.  x  9/5)  +  32  =  F. 
To  change  Fahrenheit  to  Centigrade  :  (F.  —  32)  x  5/9  =  C. 
To  change  Reaumur  to  Fahrenheit :  (R.  x  9/4)  +  32  =  F. 
To  change  Fahrenheit  to  Reaumur  :  (F.  -  32)  x  4/9  =  R. 
To  change  Centigrade  to  Reaumur  :  C.  x  4/5  =  R. 
To  change  Reaumur  to  Centigrade  :  R.  x  5/4  =  C. 

CONVERSION  FACTORS 
METRIC  TO  ENGLISH,   ENGLISH  TO  METRIC 

Meter  =  39.3704  inches 

Millimeter  =  0.03937  inches  (0.04  approximately) 

Inch  =  25.3997  mm.  (25.4  approximately) 

Liter  =  2.11  pints  (1  quart  approximately) 

Cubic  centimeter  =  16.23  minims 

Fluid  ounce  =29.578  cubic  centimeters  (30  cc.  approximately) 

Gram  =  15.432  grains 

Kilogram  =  2.204  avoirdupois  pounds 

Ounce  avoirdupois  =  28.349  grams 

Pound  avoirdupois  =  453.584  grams 

Ounce  troy  =  31.103  grams 

Square  centimeter  =  0.1548  square  inches 

Square  inch  =  6  square  centimeters  (approximately) 

Cubic  centimeter  =  0.0609  cubic  inches 

Cubic  inch  =  16  cubic  centimeters  (approximately) 


138 


APPENDIX  D 
APPAKATUS  FOB  EACH  STUDENT 

1  Erlenmeyer  flask,  1000  cc. 

2  Erlenmeyer  flasks,  600  cc. 
'2  Krlenmeyer  flasks,  300  cc. 
10  Erlenmeyer  flasks,  150  cc. 
200  test  tubes  (15  x  120  mm.) 

30  Petri  dishes  (90  mm.  diameter) 

10  glass  tumblers 

5  1-cc.  pipettes,  graduated  in  tenths  of  a  cc.' 

5  1-cc.  pipettes  (volume) 

1  5-cc.  pipette  (volume) 

1  pipette  case 

8  stain  bottles  in  tray 

3  wire  baskets  (4x4x4  inches) 
1  Bunsen  burner  and  tubing 

1  thermometer  (0°-100°C.) 

2  glass  rods  for  platinum  needles 
2  stirring  rods 

1  test-tube  cleaner 
1  rice  cooker 
1  ring  stand,  2  rings 
1  burette  clamp 

1  tripod 

2  4-in.  funnels 
1  3-in.  casserole 

1  100-cc.  graduated  cylinder 
1  copper  cup 

10  cm.  rubber  tubing  with  glass  tip 
1  pinchcock 

1  piece  wire  gauze  12  cm.  square 
1  piece  Russia  iron  12  cm.  square  • 

\  ounce  cover  glasses  18  mm.  square,  0.17  mm.  thick  (No.  2) 
50  glass  slides  25  x  75  mm. 
1  pair  cover-glass  forceps 
1  pair  fine-pointed  forceps 
1  hollow-ground  slide 
140  mm.  No.  27  platinum  wire 
139 


GLOSSARY  OF  TERMS 


amoeboid,  assuming  various  shapes 
like  an  amoeba. 

amorphous,  without  visible  differ- 
entiation in  structure. 

arborescent,  a  branched,  tree-like 
growth. 

beaded,  in  stab  or  stroke,  dis- 
jointed or  semiconfluent  col- 
onies along  the  line  of 
inoculation. 

brittle,  growth  dry,  friable  un- 
der the  platinum  needle. 

bullate,  growth  rising  in  convex 
prominences,  like  a  blistered 
surface. 

butyrous,  growth  of  a  butter-like 
consistency. 

chains,  short  chains,  composed  of 
2-8 elements;  long  chains, com- 
posed of  more  than  8  elements. 

ciliate,  having  fine,  hair-like  ex- 
tensions, like  cilia. 

cloudy,  said  of  fluid  cultures 
which  do  not  contain  pseudo- 
zoogloese. 

coagulation,  the  separation  of 
casein  from  wrhey  in  milk. 
This  may  take  place  quickly 
or  slowly,  and  as  the  result 
either  of  the  formation  of  an 
acid  or  of  a  lab  ferment. 

contoured,  an  irregular,  smoothly 
undulating  surface,  like  that 
of  a  relief  map. 


convex,  surface  the  segment  of  a 
circle,  but  flattened. 

coprophyl,  dung  bacteria. 

coriaceous,  growth  tough,  leath- 
ery, not  yielding  to  the  plati- 
num needle. 

crater iform,  round,  depressed,  due 
to  the  liquefaction  of  the  me- 
dium. 

cretaceous,  growth  opaque  and 
white,  chalky. 

curled,  composed  of  parallel  chains 
in  wavy  strands,  as  in  anthrax 
colonies. 

diastasic  action  (same  as  diastatic) , 
conversion  of  starch  into  water- 
soluble  substances  by  diastase. 

echinulate,  in  agar  stroke  a 
growth  along  line  of  inocula- 
tion, with  toothed  or  pointed 
margins  ;  in  stab  cultures, 
growth  beset  with  pointed  out- 
growths. 

effuse,  growth  thin,  veily,  unu- 
sually spreading. 

entire,  smooth,  having  a  margin 
destitute  of  teeth  or  notches. 

erose,  border  irregularly  toothed. 

filamentous,  growth  composed  of 
long,  irregularly  placed  or  in- 
terwoven filaments. 

filiform,  in  stroke  or  stab  cultures 
a  uniform  growth  along  line 
of  inoculation. 


140 


GLOSSARY  OF  TERMS 


141 


fimbriate,  border  fringed  with 
slender  processes,  larger  than 
filaments. 

floccose,  growth  composed  of  short 
curved  chains,  variously  ori- 
ented. 

flocculent,  said  of  fluids  which 
contain  pseudozooglojae,  i.e. 
small  adherent  masses  of  bac- 
teria of  various  shapes  and 
floating  in  the  culture  fluid. 

fluorescent,  having  one  color  by 
transmitted  light  and  another 
by  reflected  light. 

grumose,  clotted. 

infundibuliform,  form  of  a  funnel 
or  inverted  cone. 

iridescent,  like  mother-of-pearl. 
The  effect  of  very  thin  films. 

lacerate,  having  the  margin  cut 
into  irregular  segments  as  if 
torn. 

lobate,  border  deeply  undulate, 
producing  lobes  (see  undu- 
late). 

maximum  temperature,  tempera- 
ture above  which  growth  does 
not  take  place. 

membranous,  growth  thin,  co- 
herent, like  a  membrane. 

minimum  temperature,  tempera- 
ture below  which  growth  does 
not  take  place. 

mycelioid,  colonies  having  the 
radiately  filamentous  appear- 
ance of  mold  colonies. 

napiform,  liquefaction  with  the 
form  of  a  turnip. 

nitrogen  requirements,  the  neces- 
sary nitrogenous  food.  This 
is  determined  by  adding  to 


nitrogen-free  media  the  nitrogen 
compound  to  be  tested. 

opalescent,  resembling  the  color 
of  an  opal. 

optimum  temperature,  tempera- 
ture at  which  growth  is  most 
rapid. 

pellicle,  bacterial  growth  either 
forming  a  continuous  or  an 
interrupted  sheet  over  a  fluid. 

peptonized,  said  of  curds  dissolved 
by  trypsin. 

persistent,  lasting  many  weeks  or 
months. 

plumose,  a  fleecy  or  feathery 
growth. 

pseudozoogloeae,  clumps  of  bac- 
teria, not  dissolving  readily  in 
water,  arising  from  imperfect 
separation  or  more  or  less 
fusion  of  the  components,  but 
not  having  the  degree  of  com- 
pactness and  gelatinization 
seen  in  zoogloeae. 

pulvinate,  in  the  form  of  a  cushion , 
decidedly  convex. 

punctiform,  very  minute  colo- 
nies, at  the  limit  of  natural 
vision. 

raised,  growth  thick,  with  abrupt 
or  terraced  edges. 

rhizoid,  growth  of  an  irregular 
branched"  or  root-like  char- 
acter, as  in  B.  inycoides. 

ring  (same  as  rim),  growth  at  the 
upper  margin  of  a  liquid  cul- 
ture, adhering  more  or  less 
closely  to  the  glass. 

rapid,  developing  in  from  twenty- 
four  to  forty -eight  hours. 

repand,  wrinkled. 


142 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


saccate,  liquefaction  the  shape  of 
an  elongated  sack,  tubular, 
cylindrical. 

scum,  floating  islands  of  bacteria ; 
an  interrupted  pellicle  or  bac- 
terial membrane. 

sporangia,  cells  containing  endo- 
spores. 

spreading,  growth  extending  much 
beyond  the  line  of  inoculation, 
i.e.  several  millimeters  or  more. 

stratiform,  liquefying  to  the  walls 
of  the  tube  at  the  top  and  then 
proceeding  downwards  hori- 
zontally. 

transient,  a  few  days. 

turbid,  cloudy  with  flocculent  par- 
ticles ;  cloudy  plus  flocculence. 

umbonate,  having  a  button-like, 
raised  center. 

undulate,  border  wavy,  with  shal- 
low sinuses. 


vermiform-contoured,  growth  like 
a  mass  of  worms,  or  intestinal 
coils. 

verrucose,  growth  wart-like  with 
wart-like  prominences. 

villous,  growth  beset  with  hair- 
like  extensions. 

viscid,  growth  follows  the  needle 
when  touched  and  withdrawn ; 
sediment  on  shaking  rises  as  a 
coherent  swirl. 

zooglceae,  firm  gelatinous  masses 
of  bacteria,  one  of  the  most 
typical  examples  of  which  is  the 
Streptococcus  mesenterioides  of 
sugar  vats  (Leuconostoc  mesente- 
rioides),  the  bacterial  chains 
being  surrounded  by  an  enor- 
mously thickened  firm  cover- 
ing, inside  of  which  there  may 
be  one  or  many  groups  of  the 
bacteria. 


TEXT-BOOKS  ON  GENERAL  AND  DAIRY 
BACTERIOLOGY 


CHESTER,  A  Manual  of  Determinative  Bacteriology.   The  Macmillan 

Company,  New  York,  1901. 

CONN,  Bacteria  in  Milk.    Orange  Judd  Co.,  New  York,  1907. 
FISCHEK,  Structure  and  Functions  of  Bacteria.    Clarendon  Press, 

New  York,  1900. 
FROST,  Laboratory  Bacteriology.    The  Macmillan  Company,  New 

York,  1903. 
JENSEN,  Essentials  of  Milk  Hygiene.    Translation  by  Pearson.  J.  B. 

Lippincott  Company,  Philadelphia,  1907. 
LAFAR,  Handbuch  der  Technischen   Mykologie.    Second  Edition. 

Gustav  Fischer,  Jena,  1904-1909. 
Li. 1 1 MANN  and    XKTMAXN,  Atlas  and  Principles  of  Bacteriology. 

W.  B.  Saunders  &  Company,  Philadelphia,  1901. 
"Milk  and  its  Relation  to  Public  Health  "  (by  various  authors), 

Bulletin  1^1,  Hygienic  Laboratory.    United  States  Public  Health 

and  Marine  Hospital  Service,  Washington,  1908. 
Me  IK    and    RITCHIE,    Manual   of   Bacteriology.    Fourth   Edition. 

Henry  Froude,  and  Hodder  &  Stoughton,  Edinburgh,  1907. 
PRESCOTT  and  WINSLOW,  Elements  of  Water  Bacteriology.    Second 

Edition.    Wiley  &  Sons,  New  York,  1908. 

"Report  of  the  Committee  on  Standard  Methods  of  Water  Anal- 
ysis to  the  Laboratory  Section  of  the  American  Public  Health 

Association."    Journal  of  Infectious  Diseases,  Supplement  No.  I, 

Chicago,  1905. 
HUSSELL,  Outlines  of  Dairy  Bacteriology.    Eighth  Edition.    H.  L. 

Russell,  Madison,  Wisconsin,  1907. 
SVVITIIKXBANK  and  NEWMAN,  Bacteriology  of  Milk.    John  Murray, 

London, 1903. 


143 


INDEX 


Abbe  condenser,  39 

Acid-fast  bacteria,  1 13 

A  gar,  5  ;  filtering  of,  7,  15  ;  prepa- 
ration of,  15 

Alcohol,  test  for,  79 

Ammonia,  test  for,  71 

Anaerobic  bacteria,  cultivation  of, 
82 

Aniline-water  gentian  violet,  43 

Animals,  inoculation  of,  114  ;  post- 
mortem examination  of,  114 

Antagonism,  20 

Autoclave,  10 

Bacillus  coli  communis,  18,  70,  77, 
97,  106 

Bacillus  fluorescens  liquefaciens, 
97,  102 

Bacillus  lactis  acidi,  77,  97,  106 

Bacillus  lactis  aerogenes,  77 ,97, 106 

Bacillus  lactis  longi,  80 

Bacillus  lactis  viscosus,  79 

Bacillus  mesentericus  ruber,  74 

Bacillus  mesentericus  vulgatus,  79 

Bacteria,  direct  enumeration  of, 
122  ;  effect  of  creaming  on  dis- 
tribution of,  93  ;  examination  of 
living,  49 ;  measuring  of,  51  ; 
preliminary  cultivation  of,  65 ; 
rdle  of,  in  cheese  ripening,  104 

Bacterial  species,  identification  of, 
66 

Beef  extract,  5,  16 

Blood-counting  cell,  118 

Books,  reference,  145 

Bouillon,  12 

Broth,  preparation  of,  12 

Brownian  movement,  50 

Burri  culture  tube,  84 

Butter,  analysis  of,  95;  flavor  of, 
95 ;  keeping  quality  of,  101 


Canada  balsam,  45 

Capsule  stain,  48 

Carbohydrates,  action  of  bacteria 

on,  67 

Carbol-fuchsin,  43 
Cheese,  analysis  of,  105 ;  ripening 

of,  103 ;  sampling  of,  105 
Chromogenesis,  74 
Colonies,  study  of,  32 
Commercial  starters,  97 
Cotton  plugs,  9 

Cover  glasses,  41 ;  cleaning  of,  42 
Cream,  examination  of,  94 
Cultures,    incubation    of,    23,   24; 

testing  purity  of,  31 

Diaphragm,  iris,  39 
Diphtheria,  111 

Enzymes,  104 

Fermentation,  acid,  76;  alcoholic, 
78 ;  bitter,  80 ;  butyric,  81 ;  cycle 
of,  80 ;  sweet-curdling,  77 

Fermentation  tests,  107 

Fibrin,  staining  of,  122 

Fishing,  33 

Flagella,  staining  of,  47 

Fuchsin,  43 

Galactase,  104 

Gas,  analysis  of,  69 ;  measurement 
of,  69 ;  production  of,  68 

Gasometer,  Frost's,  68 

Gelatin,  5  ;  effect  of  heat  on,  14 ; 
filtering  of,  7;  preparation  of, 
14 

Gentian  violet,  43 

Gerber  fermentation  test,  107 

Glassware,  1 ;  cleaning  of,  2 ;  ster- 
ilization of,  3 


145 


146 


EXPERIMENTAL  DAIRY  BACTERIOLOGY 


Gram's  stain,  43 
Gram's  solution,  47 

Hanging  block,  51 
Hanging  drop,  49 
Heat,  effect  on  milk,  89 
Heated  milk,  detection  of,  91 
Homogeneous  milk,  90 

Incubation,    temperature    of,    23; 

time  of,  23 
Indol,  test  for,  72 
Inhibition,  20 
Iodine  solution,  43 
lodoform,  79 

Leucocytes,  116,  117 

Light,  39 

Litmus,  5 ;  preparation  of,  18 

Litmus  lactose  agar,  29 

Litmus  lactose  gelatin,  29 

Litmus  milk,  17 

Lugol's  solution,  43 

Manure,  contamination  of  milk 
from,  57 

Meat  infusion,  5 

Media,  care  of,  11 ;  clearing  of,  16; 
filtering  of,  7  ;  ingredients  of,  5 ; 
neutralization  of,  6  ;  reaction  of, 
5;  sterility  of,  11,  17;  steriliza- 
tion of,  9 ;  sugar-free,  18 

Methylene  blue,  43 

Micrometer,  ocular,  51  ;  stage,  52 

Micron,  52 

Microscope,  37  ;  accessories  of,  37 

Milk,  acid  fermentation  of,  76 ; 
acidity  of ,  16 ;  agar,  75 ;  alcoholic 
fermentation  of,  78  ;  analysis  of, 
19,  29;  bitter  fermentation  of, 
80 ;  butyric  fermentation  of,  81 ; 
condensed,  88 ;  contamination  of, 
53 ;  cycle  of  fermentations  in,  80 ; 
determination  of  acidity  of,  76 ; 
digestion  of,  77  ;  dilution  of,  21 ; 
examination  of :  for  diphtheria, 
112;  for  fibrin,  122;  for  leu- 
cocytes, 117,  120;  for  pyogenic 
bacteria,  114,  124;  for  tubercu- 
losis, 112,  114  ;  for  typhoid,  112  ; 
homogeneous,  90;  hygiene  of, 
110;  microscopical  examination 


of,  116;  pasteurization  of,  89; 
preservation  of,  86 ;  ropy,  79  ; 
slimy,  79;  sweet-curdling  of,  77 

Needles,  30 ;  Ravenel's,  31 ;  sterili- 
zation of,  31 
Nessler  solution,  71 
Nitrites,  test  for,  71 

Object  slides,  41  ;  cleaning  of,  41 
Oidium  lactis,  81 
Oil-immersion  objective,  40 
Oxygen,  relation  of  bacteria  to,  72 

Pasteurization,  89 

Pedesis,  50 

Pepsin,  104 

Peptone,  5 ;  solution  of,  72 

Physiological  salt  solution,  18 

Pipettes,  calibration  of,  125  ;  prep- 
aration of,  125  ;  sterilization  of,  4 

Plate  cultures,  31 ;  counting  of,  22  ; 
incubation  of,  23  ;  study  of,  32 

Preservatives,  87 

Pure  cultures,  isolation  of,  30 

Pus  cells,  116 

Qualitative  analysis,  29 

Quantitative  analysis,  19,  26  ;  accu- 
racy of,  26 ;  comparative  method, 
28 ;  expression  of  results,  25 

Rennet,  104 
Ropy  milk,  48 

Sanitary  milk,  plan  for  preparation 
of,  63 

Separator  slime,  examination  of, 
94 

Shake  cultures,  69 

Spore  stain,  48 

Spores,  test  for,  73 

Stab  cultures,  34  ;  study  of,  35 

Stained  preparations,  examination 
of,  46  ;  making  of,  44  ;  mounting 
of,  45 

Staining  solutions,  42 

Staphlococcus  pyogenes  aureus,  116 

Starters,  97 ;  flavor-producing  prop- 
erties of,  100 ;  preservation  of 
properties  of,  100 ;  purity  of,  97  ; 
vitality  of,  99 


INDEX 


147 


Sterilization   by   dry  heat,    3;   by 

moist  heat,  9 
Storch's  reaction,  91 
Strainers,  61 

Streak  cultures,  study  of,  35 
Streptococcus  Hollandicus,  77 
Streptococcus  pyogenes,  115,  116, 

124 
Sugars,   5;  effect  of  heat  on,  17; 

media  free  from,  70 

Temperature,  effect  of,  on  bacterial 
growth,  88 ;  relation  of  bacteria 
to,  73 

Test-tube  cultures,  33  ;  study  of,  35 

Thermal  death,point,  87 


Thermometer  scales,  138 
Tuberculosis,  110 
Tubes,  plugging  of,  9 
Typhoid  fever,  111 

Udder,  bacteria  from,  58 ;  infection 

of,  59 
Utensils,   contamination    of    milk 

from,  60 ;  washing  of,  63 

Wash  water,  effect  of,  on  butter, 

101 ;  testing  of,  102 
Water  blanks,  preparation  of,  18 
Wisconsin  Curd  Test,  107 

Yeasts,  isolation  of,  78 


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